Method for body fluid identification
阅读说明：本技术 用于体液鉴定的方法 (Method for body fluid identification ) 是由 帕特里夏·阿尔瓦尼 拉谢尔·弗莱明 杰施里·帕特尔 于 2018-10-02 设计创作，主要内容包括：犯罪现场调查人员需要鉴定生物学组织或体液类型。这样的分析通常使用常规化学、血清学和酶学测试进行以鉴定体液或组织,然而,这些测试可能不可靠,并且常常不能满足法医学分析所需的特异性和灵敏度。本发明提供了通过检测特定RNA序列来准确鉴定循环血液、唾液、精子、精液、月经液和阴道物质的方法。特别地,本发明提供了用于确定生物学样品的类型的方法,其包括以下步骤：从所述样品中检测与HBD、SLC4A1、GYPA、FDCSP、HTN3、STATH、PRM1、TNP1、PRM2、KLK2、MSMB、TGM4、MMP10、STC1、MMP3、MMP11、CYP2B7P、格氏乳杆菌(Lactobacillus gasseri)(L.gass)和卷曲乳杆菌(Lactobacillus crispatus)(L.crisp)中的任一种或更多种相关的RNA,以及确定所述样品是否是循环血液、唾液、精子、精液、月经液或阴道物质。(Criminal scene investigators need to identify biological tissue or fluid types. Such assays are typically performed using conventional chemical, serological and enzymatic tests to identify body fluids or tissues, however, these tests may be unreliable and often fail to meet the specificity and sensitivity required for forensic analysis. The present invention provides methods for accurately identifying circulating blood, saliva, sperm, semen, menstrual fluid, and vaginal material by detecting specific RNA sequences. In particular, the present invention provides a method for determining the type of a biological sample comprising the steps of: detecting RNA associated with any one or more of HBD, SLC4A1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, Lactobacillus gasseri (L.gass) and Lactobacillus crispatus (L.crisps) from the sample, and determining whether the sample is circulating blood, saliva, sperm, semen, menstrual fluid or vaginal material.)
1. A method for determining the type of a biological sample, comprising the steps of: detecting RNA associated with any one or more of HBD, SLC4A1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, Lactobacillus gasseri (L.gass) and Lactobacillus crispatus (L.crisps) from the sample, and determining whether the sample is circulating blood, saliva, sperm, semen, menstrual fluid or vaginal material.
2. The method of claim 1, comprising detecting a mutation in a nucleotide sequence that is identical to SEQ ID No: 1 to 19.
3. The method of claim 1 or 2, wherein the step of detecting the RNA comprises using one or more primers specific for any one or more of HBD, SLC4a1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, lactobacillus gass (l.gass), and lactobacillus crispatus (l.crispp).
4. The method of claim 3, wherein the one or more primers are selected from the group consisting of SEQ ID Nos: 20 to 57.
5. The method of any one of claims 1 to 4, comprising determining whether the biological sample is circulating blood, comprising the steps of: using SEQ ID No: 20 and 21, and/or detecting HBD-associated RNA using the primers of SEQ id no: 22 and 23, and/or detecting RNA associated with SLC4a1 using the primers of SEQ ID nos: primers 24 and 25 detect RNA associated with GYPA.
6. The method of any one of claims 1 to 4, comprising determining whether the biological sample is saliva, comprising the steps of: using SEQ ID No: 26 and 27, and/or detecting RNA associated with FDCSP using the primers of SEQ id no: 28 and 29, and/or using the primers of SEQ ID nos: primers 30 and 31 detect RNA associated with STATH.
7. The method of any one of claims 1 to 4, comprising determining whether the biological sample is sperm, comprising the steps of: using SEQ ID No: 32 and 33, and/or detecting RNA associated with PRM1 using the primers of SEQ id no: 34 and 35, and/or detecting RNA associated with TNP1 using the primers of SEQ ID nos: primers 36 and 37 detected RNA associated with PRM 2.
8. The method according to any one of claims 1 to 4, comprising determining whether the biological sample is semen, comprising the steps of: using SEQ ID No: 38 and 39, and/or using the primers of SEQ id no: 40 and 41, and/or using the primers of SEQ ID nos: primers 42 and 43 detect RNA associated with TGM 4.
9. The method of any one of claims 1 to 4, comprising determining whether the biological sample is menstrual fluid, comprising the steps of: using SEQ ID No: 44 and 45, and/or using the primers of SEQ ID nos: 46 and 47, and/or using the primers of SEQ ID nos: 48 and 49, and/or using SEQ ID No: primers 50 and 51 detect RNA associated with MMP 11.
10. The method of any one of claims 1 to 4, comprising determining whether the biological sample is vaginal material, comprising the steps of: using SEQ ID No: 52 and 53, and/or using the primers of SEQ ID nos: 54 and 55, and/or using the primers of SEQ ID nos: primers 56 and 57 detect RNA associated with l.crisps.
11. The method of any one of claims 1 to 10, comprising testing the biological sample for the presence of RNA in all of HBD, SLC4a1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, lactobacillus gass (l.gass), and lactobacillus crispatus (l.crispp).
12. The method of any one of claims 1 to 11, comprising detecting HTN3 and FDCSP; and/or SLC4a1, HBD, STC1 and MMP10 and/or TNP1, PRM1, KLK2, MSMB and CYP2B 7.
13. The method of any one of claims 1 to 12, wherein the primer is labeled.
14. The method of claim 13, wherein the primer is labeled with a fluorescent label, biotin, radioactive or non-radioactive label.
15. The method of any one of claims 1 to 14, wherein the RNA is detected using an amplification method.
16. The method of claim 15, wherein the amplification method is selected from the group comprising: polymerase Chain Reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative reverse transcriptase PCR (qRT-PCR), multiplex PCR, multiplex ligation dependent probe amplification (MLPA) or quantitative PCR (Q-PCR).
17. A kit for use in the method of any one of claims 1 to 16, the kit comprising at least one primer pair selected from the group consisting of: SEQ ID No: 20 and 21, 22 and 23, 24 and 25, 26 and 27, 28 and 29, 30 and 31, 32 and 33, 34 and 35, 36 and 37, 38 and 39, 40 and 41, 42 and 43, 44 and 45, 46 and 47, 48 and 49, 50 and 51, 52 and 53, 54 and 55, and 56 and 57.
Technical Field
The field of technology is the detection of RNA sequences and the use of these sequences for the identification and typing of samples, in particular samples containing degraded RNA.
Background
In many cases, a crime scene investigator will encounter a cell or fluid of interest, but will need to determine what tissue or fluid it is. This information can be important for establishing an active scenario for the case. For example, the presence of menstrual blood may indicate sexual activity, while circulating blood may be the result of trauma. Such assays are typically performed using conventional chemical, serological and enzymatic tests to identify body fluids or tissues, however, these tests may be unreliable and often fail to meet the specificity and sensitivity required for forensic analysis.
Messenger RNA (mRNA) analysis based on unique gene expression patterns in cells and tissues has become a method to overcome these limitations [1-4 ]. Co-extraction of DNA/RNA for combined Short Tandem Repeat (STR) and body fluid analysis has now become an effective and comprehensive tool used in case research laboratories around the world. However, since 2003 the introduction of differentially expressed mRNA for forensic saliva analysis [2], only a small fraction of the "core" markers were used for multiplex design. These include histone 3(HTN3) and sialoprotein (statherin) (STATH) of saliva and oral mucosa [1, 3, 5-7],
The ability to accurately detect and quantify RNA abundance is a fundamental capability of molecular biology. A wide variety of RNA detection methods are currently available, ranging from non-amplification methods (in situ hybridization, microarray and NanoString nCounter) to amplification (PCR) -based methods (reverse transcriptase PCR (RT-PCR) and quantitative reverse transcriptase PCR (qRT-PCR)). With the exception of RNAseq (next generation sequencing, also known as second generation sequencing or massively parallel sequencing), a key prerequisite for all RNA detection technologies is the existing knowledge of the target RNA sequence. This targeting is aided by the oligonucleotide sequence in both non-amplification methods (probes) and amplification-based methods (primers).
Methods for PCR primer design have been developed [1, 2], but are still based on the core criteria of specificity, thermodynamics, secondary structure, dimerization and amplicon length [3-7 ]. In addition to these criteria, RT-PCR primer design (for RNA amplification) also takes into account exon boundary coverage to ensure that only cDNA is amplified and to avoid amplification of genomic DNA [8 ]. Among other experimental factors [9-14], PCR primer design is widely regarded as important for target amplification, detection and quantification [3, 8, 11, 15-18 ].
Although improved primer design can improve performance, target molecules must also be considered. RNA is unstable and readily degraded [19-22 ]. Conventional methods recommend that the sample RNA Integrity (RIN) be at least RIN8 or higher to ensure proper performance [23-26 ]. RIN values range from 10 (intact) to 1 (fully degraded). The gradual degradation of RNA is reflected by a continuous movement towards shorter RNA fragments, the shorter the RNA fragments the more RNA is degraded. In this case, shorter means that the RNA fragments are not as long as the undegraded RNA and, over time, the RNA fragments break into smaller and smaller fragments.
Furthermore, in the case of forensic, clinical, FFPE and environmental samples, which must be analyzed for real-life, some degradation is inevitable. The adverse effects of RNA degradation on RNA detection and quantification have been well documented [24, 27-30 ]. There is currently no clear solution to this problem, except to avoid analyzing degraded RNA.
Here, the inventors have established a method for accurately identifying circulating blood, saliva, sperm, semen, menstrual fluid and vaginal substances by detecting specific RNA sequences.
It is an object of the present invention to provide methods and/or materials for the specific detection of tissue types in unknown samples and/or to at least provide the public with a useful choice.
Disclosure of Invention
Sample typing
In a first aspect, the present invention provides a method of typing a sample, the method comprising the step of detecting an RNA sequence in the sample by the method of the invention, wherein the detection of the RNA sequence marker indicates the type of the sample.
The method may involve typing the sample using only one pair of primers or a single probe. Alternatively, multiple pairs of primers or multiple probes may be used.
In particular, the present invention provides a method for determining the type of a biological sample comprising the steps of: detecting RNA associated with any one or more of HBD, SLC4A1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, L.gass and L.crisp from said sample and determining whether said sample is circulating blood, saliva, sperm, semen, menstrual fluid or vaginal material.
The method comprises detecting whether the biological sample is circulating blood, comprising the step of detecting RNA associated with HBD, SLC4a1, and/or GYPA.
The method comprises detecting whether the biological sample is saliva, comprising the step of detecting RNA associated with FDCSP and/or HTN3 and/or STATH.
The method comprises detecting whether the biological sample is sperm, comprising the step of detecting RNA associated with PRM1, TNP1, and/or
The method comprises the step of detecting whether the biological sample is semen, including the step of detecting RNA associated with KLK2, MSMB and/or TGM 4.
The method includes detecting whether the biological sample is menstrual fluid, including the step of detecting RNA associated with MMP10 and/or STC1 and/or MMP3 and/or MMP 11.
The method comprises detecting whether the biological sample is vaginal material comprising the step of detecting RNA associated with CYP2B7P, l.gass and/or l.crisp.
The methods of the invention include, but are not limited to, the use of multiplex PCR.
Sample typing by multiplex PCR
In one embodiment, multiplex PCR is performed with one or more primers, at least one of which is diagnostic for the type of sample.
Preferably, the method comprises the use of one or more primers specific for any one of HBD, SLC4a1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, l.gass or l.crisp, more preferably, the primers are selected from the group consisting of SEQ ID No: any one of 20 to 57.
The method comprises detecting whether the biological sample is circulating blood, comprising the steps of: using SEQ ID No: 20 and 21, and/or using the primers of SEQ ID No: 22 and 23, and/or detecting RNA associated with SLC4a1 using the primers of SEQ ID nos: primers 24 and 25 detect RNA associated with GYPA.
The method comprises detecting whether the biological sample is saliva, comprising the steps of: using SEQ ID No: 26 and 27, and/or detecting RNA associated with FDCSP using the primers of SEQ ID nos: 28 and 29, and/or using the primers of SEQ ID nos: primers 30 and 31 detect RNA associated with STATH.
The method comprises detecting whether the biological sample is sperm, comprising the steps of: using SEQ ID No: 32 and 33, and/or detecting RNA associated with PRM1 using the primers of SEQ ID No: 34 and 35, and/or detecting RNA associated with TNP1 using the primers of SEQ ID nos: primers 36 and 37 detected RNA associated with
The method comprises the step of detecting whether the biological sample is semen or not, and comprises the following steps: using SEQ ID No: 38 and 39, and/or using SEQ ID No: 40 and 41, and/or using the primers of SEQ ID nos: primers 42 and 43 detect RNA associated with TGM 4.
The method comprises detecting whether the biological sample is menstrual fluid, comprising the steps of: using SEQ ID No: 44 and 45, and/or using the primers of SEQ ID nos: 46 and 47, and/or using the primers of SEQ ID nos: 48 and 49, and/or using seq id nos: primers 50 and 51 detect RNA associated with MMP 11.
The method comprises detecting whether the biological sample is vaginal material, comprising the steps of: using SEQ ID No: 52 and 53, and/or using the primers of SEQ ID nos: 54 and 55, and/or using the primers of SEQ ID nos: primers 56 and 57 detect RNA associated with l.crisps.
Primer and method for producing the same
In another embodiment, the invention provides a primer capable of hybridizing to a stabilizing region of an RNA sequence or a cDNA corresponding to a stabilizing region or its complement.
In another embodiment, the invention provides a primer comprising a sequence identical to SEQ ID NO: 1 to 19 or any portion of its complement, having at least 70% identity.
In another embodiment, the primer consists of a sequence identical to SEQ ID NO: 1 to 19 or the complement thereof, having a sequence composition of at least 5 nucleotides with at least 70% identity.
In another embodiment, the primer comprises SEQ ID NO: 1 to 19 or a complement thereof.
In another embodiment, the primer consists of SEQ ID NO: 1 to 19 or the complement thereof, or a sequence of at least 5 nucleotides of the sequence of any one of 1 to 19.
In another embodiment, the primer comprises a sequence selected from the group consisting of SEQ ID NOs: 20 to SEQ ID NO: 57 or a complement of any one thereof.
In another embodiment, the primer consists of a sequence selected from the group consisting of SEQ ID NOs: 20 to SEQ ID NO: 57 or a complement of any one thereof.
In another embodiment, the primer is selected from the group consisting of SEQ ID NOs: 20 to SEQ ID NO: 57 or a complement of any one thereof.
In another embodiment, the primer comprises an attached label or tag.
In another embodiment, the tag or label primer does not occur in nature.
The primers of the invention can be used in microarrays or chips or similar products for the detection of RNA sequences.
Primer kit
In another embodiment, the invention provides a kit comprising at least one primer of the invention.
Preferably, the kit comprises at least one primer pair selected from the group consisting of: SEQ ID No: 20 and 21, 22 and 23, 24 and 25, 26 and 27, 28 and 29, 30 and 31, 32 and 33, 34 and 35, 36 and 37, 38 and 39, 40 and 41, 42 and 43, 44 and 45, 46 and 47, 48 and 49, 50 and 51, 52 and 53, 54 and 55, and 56 and 57.
In one embodiment, the kit further comprises instructions for use.
Probe needle
In another embodiment, the invention provides probes capable of hybridizing to an RNA sequence or a corresponding cDNA or its complement. Preferably, the probe is capable of hybridizing to any one of HBD, SLC4a1, GYPA, FDCSP, HTN3, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, CYP2B7P, l.gass and l.crisp.
In another embodiment, the invention provides a probe comprising a nucleotide sequence identical to SEQ ID NO: 1 to 19 or any portion of its complement, having at least 70% identity.
In another embodiment, the probe consists of a nucleotide sequence identical to SEQ ID NO: 1 to 19 or the complement thereof, having a sequence composition of at least 10 nucleotides with at least 70% identity.
In another embodiment, the probe comprises SEQ ID NO: 1 to 19 or a complement thereof, or a sequence of at least 10 nucleotides of the sequence of any one of 1 to 19.
In another embodiment, the probe consists of SEQ ID NO: 1 to 19 or the complement thereof, or a sequence of at least 10 nucleotides of the sequence of any one of 1 to 19.
In another embodiment, the probe comprises a linked label or tag.
In another embodiment, the label or tagged probe does not occur in nature.
The primers of the invention can be used in microarrays or chips or similar products for the detection of RNA sequences.
Probe kit
In another embodiment, the invention provides a kit comprising at least one probe of the invention.
Preferably, the kit comprises at least 2, more preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20, more preferably at least 21, more preferably at least 22, more preferably at least 23, more preferably at least 24, more preferably at least 25, more preferably at least 26, more preferably at least 27, more preferably at least 28, more preferably at least 29, more preferably at least 30, more preferably at least 31, more preferably at least 32 probes, more preferably at least 33 probes, more preferably at least 34, more preferably at least 35, more preferably at least 36, more preferably at least 37, more preferably at least 30 probes of the invention.
In one embodiment, the kit further comprises instructions for use.
Microarray
In another aspect, the invention provides a microarray comprising a nucleic acid sequence identical to SEQ ID NO: 1 to SEQ ID NO: 19 or any portion of its complement, having at least 70% identity thereto.
In another aspect, the invention provides a microarray comprising SEQ ID NO: 1 to SEQ ID NO: 19 or a complement thereof, or a sequence of at least 5 nucleotides of the sequence of any one of.
In another aspect, the invention provides a microarray comprising a nucleic acid sequence identical to SEQ ID NO: 1 to SEQ ID NO: 19 or any portion of its complement, a sequence of at least 10 nucleotides of a sequence having at least 70% identity.
In another aspect, the invention provides a microarray comprising SEQ ID NO: 1 to SEQ ID NO: 95 or a complement thereof, or a sequence of at least 19 nucleotides of the sequence of any one of them.
Preferably, the sequence comprises the following nucleotides of the sequence of the invention: at least 5, more preferably at least 10, more preferably at least 15, more preferably at least 20, more preferably at least 25, more preferably at least 30, more preferably at least 35, more preferably at least 40, more preferably at least 45, more preferably at least 50, more preferably at least 55, more preferably at least 60, more preferably at least 65, more preferably at least 70, more preferably at least 75, more preferably at least 80, more preferably at least 85, more preferably at least 90, more preferably at least 95, more preferably at least 100, more preferably at least 120, more preferably at least 140, more preferably at least 160, more preferably at least 180, more preferably at least 200, more preferably at least 240, more preferably at least 250.
One skilled in the art would understand how to select appropriate probes or primers to detect any of the listed markers based on the information in the sequence listing and elsewhere in the specification.
One skilled in the art understands that probes or primers can be generated that can hybridize to any portion of the stabilizing region. The probes and primers mentioned herein are given as examples only to demonstrate that the stabilizing region can be used for identification and typing of degraded RNA. Any primer or probe complementary to the stabilizing region is suitable for use in the methods of the invention.
Accordingly, the present invention provides:
1. a method for determining the type of a biological sample, comprising the steps of: detecting RNA associated with any one or more of HBD, SLC4A1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, Lactobacillus gass (L.gass) and Lactobacillus crispatus (L.crispp) from the sample and determining whether the sample is circulating blood, saliva, sperm, semen, menstrual fluid or vaginal material.
2. The method of 1, comprising detecting a mutation in a sequence that is identical to SEQ ID No: 1 to 19.
3. The method of 1 or 2, wherein the step of detecting the RNA comprises using one or more primers specific for any one or more of HBD, SLC4a1, GYPA, FDCSP, HTN3, STATH, PRM1, TNP1, PRM2, KLK2, MSMB, TGM4, MMP10, STC1, MMP3, MMP11, CYP2B7P, lactobacillus gass (l.gass) and lactobacillus crispatus (l.crispp).
4. The method of 3, wherein the one or more primers are selected from the group consisting of SEQ ID Nos: 20 to 57.
5. The method according to any one of
6. The method according to any one of
7. The method of any one of
8. The method according to any one of
9. The method according to any one of
10. The method according to any one of
11. The method of any one of
12. The method of any one of
13. The method of any one of
14. The method of 13, wherein the primer is labeled with a fluorescent label, biotin, radioactive or non-radioactive label.
15. The method of any one of
16. The method of claim 15, wherein the amplification method is selected from the group comprising: polymerase Chain Reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative reverse transcriptase PCR (qRT-PCR), multiplex PCR, multiplex ligation dependent probe amplification (MLPA) or quantitative PCR (Q-PCR).
17. A kit for use in the method of any one of
One skilled in the art will understand the relationship between the marker gene, the mRNA encoded by the marker gene, and the stable region within the mRNA. One skilled in the art will understand that a given sequence is a DNA sequence corresponding to an mRNA or a stable region within an mRNA.
Drawings
FIG. 1 expression patterns of HBD, SLC4A1, TNP1, KLK2, MMP3, and
FIG. 2 comparison of the sensitivity of six novel mRNAs with four well-known markers [1 ]. The upper diagram: HBD and SLC4A1 compared to GYPA, 3 samples were used, 2, 1 and 0.5. mu.L circulating blood, respectively, with a primer concentration of 0.2. mu.M. Second row from top: TNP1 compared to PRM2, 9 samples of 1 μ L semen from three donors were used with a primer concentration of 0.05 μ M. Second row from bottom: KLK2 compared to TGM4 using 3 samples, 2, 1 and 0.5. mu.L of seminal fluid (no sperm) with a primer concentration of 0.1. mu.M. The following figures: MMP3 and STC1 compared to MMP11, using 9 month menstrual fluid samples from two donors (
FIG. 3 RNA-Seq results (FPKM) for two known markers (GYPA, MMP11) and four new mRNA candidates (HBD, SLC4A1, MMP3, STC 1). BL — circulating blood; BU being oral cavity; MF ═ menstrual fluid; VM is vaginal substance.
FIG. 4 primer sequences and expected amplicon sizes for all markers included in the three multiplex assays.
FIG. 5 humoral specificity of three multiplex assays.
FIG. 6. the following electrophoretograms: A. oral samples, b. menstrual fluid samples, and c. mixed samples of semen and vaginal material. Each sample was amplified using multiplex D (top), multiplex Q (middle) and multiplex P (bottom).
FIG. 7. Effect of multiplexing. APH obtained in multiplex (white bars) and singleplex reactions (shaded) for: a.0.05 μ M FDCSP and 0.012 μ M HTN3, B.0.05 μ M HBD and 0.04 μ M SLC4A1, C.0.04 μ M MMP10 and 0.02 μ M STC1, D.0.03 μ M PRM1 and 0.04 μ M TNP1, E.0.14 μ M KLK2 and 0.03 μ M MSMB, and F.0.02 μ M MCPP 2B7P.
Figure 8 resolution of body fluid mixture. Values are given in RFU. MF was collected from natural circulation donors on
Figure 9. post-coital vaginal samples were amplified using multiplex P.
FIG. 10 marker detection in aged samples. Peak heights (RFU) were obtained from aged body fluid samples, aged RNA and aged cDNA stored or frozen at room temperature for 15 to 35 months.
FIG. 11 analysis of case type samples. The expected results are highlighted.1After the mRNA analysis is complete, the expected results are disclosed. BL ═ circulating blood, SA ═ saliva, SP ═ sperm, SF ═ semen, VM ═ vaginal material, and NR ═ no results.
2As disclosed [2]]CellTyper amplification was performed. PCR products were isolated on a Genetic Analyzer 3130xl with a peak amplitude threshold of 100 RFU.
The invention will now be illustrated by the following non-limiting examples.
Detailed Description
In this specification, reference has been made to some patent specifications, other external documents, or other sources of information, which are generally intended to provide a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
The term "comprising" as used in the specification and claims means "consisting at least in part of"; that is, when interpreting statements in this specification and claims which include "comprising," the features prefaced by that term in each statement all need to be present but other features can also be present. Related terms such as "including" and "comprising" are to be interpreted in a similar manner. However, in some preferred embodiments, inclusion may be replaced with a composition consisting of.
As used herein, the term "RNA" means messenger RNA, small RNA, microrna, non-coding RNA, long non-coding RNA, small non-coding RNA, ribosomal RNA, small nucleolar RNA, transfer RNA, and all other RNA species and sequences.
As used herein, the term "stabilizing region" refers to a region or regions in an RNA sequence that have more aligned sequencing reads than another region or regions of the same RNA sequence.
As used herein, the term "degraded RNA" refers to RNA that is no longer intact. In other words, the theoretical full-length RNA annotated or predicted in the sequence database is no longer intact. The full-length RNA may be fragmented and/or some nucleotides are no longer present. This may occur anywhere along the RNA sequence.
The inventors emphasize that it is not essential how to measure the level of RNA degradation and the invention consists in that the method is also applicable to samples where a certain degree of degraded RNA may be present.
The present inventors have determined a method of identifying the type of biological sample, with the aim that the method can be used to identify biological samples obtained in forensic situations. In particular, the method may be used to determine whether a given biological sample is circulating blood, saliva, sperm, semen, menstrual fluid or vaginal material.
The present invention includes determining the presence of RNA for markers that have been identified by the inventors as specific to circulating blood, saliva, sperm, semen, menstrual fluid, and/or vaginal material. As shown in table 1, to identify circulating blood, markers HBD and/or SLC4a1 and/or GYPA; for saliva, the markers FDCSP and/or HTN3 may be used; for sperm, the markers PRM1 and/or TNP1 and/or
It will be appreciated that a single marker or pair of markers specific for a particular type may be used to test whether a given sample is of that type. Alternatively, one specific marker or specific marker pair may be used to determine whether a given sample is of one or two or more types. The present invention may also be used to test for the presence of RNA for all markers HBD, SLC4a1, GYPA, FDCSP, HTN3, PRM1, TNP1, PRM2, KLK2, TGM4, MSMB, MMP10, STC1, MMP3, CYP2B7P, l.gass and l.crisp in a sample to determine whether the sample is circulating blood, saliva, sperm, semen, menstrual fluid and/or vaginal material.
The methods of the invention then involve generating probes or primers that target the mRNA or a stable region in the mRNA. The method allows for improved detection of such RNA sequences, particularly in samples where the RNA is or has undergone degradation.
Table 1.
1. The marker (shown) is optional
RNA degradation
Although improved primer or probe design can produce performance improvements in amplification and hybridization methods, target molecules must also be considered. RNA is unstable and readily degraded [40-43 ]. Conventional methods recommend that the sample RNA Integrity (RIN) be at least RIN8 or higher to ensure proper performance [44-47 ].
Other measures of RNA sequence degradation are known, for example DV200[63 ].
However, the skilled person understands that it is not essential how to measure the level of RNA degradation, and the present invention resides in the ability to detect degraded RNA.
In the case of real-life samples that must be analyzed, such as forensic, clinical, formalin-fixed paraffin-embedded (FFPE) and environmental samples, some degradation is inevitable. The adverse effects of RNA degradation on RNA detection and quantification have been well documented [45, 48-51 ].
The methods and materials of the invention enable improved detection of an RNA sequence of interest, particularly when the RNA sample has been degraded. This enables typing of samples comprising degraded RNA, including samples with RIN values less than 8. This was particularly unexpected because prior to the present invention it was generally believed that detection and typing of degraded RNA sequences with RIN less than 8 could not be achieved with acceptable performance values.
RIN values range from 10 (intact) to 1 (fully degraded). The gradual degradation of RNA is reflected by a continuous movement towards shorter RNA fragments, the shorter the RNA fragments the more RNA is degraded. When the RIN value is less than 1, this indicates that RNA degradation is beyond detection.
The inventors have found that although the probes and primers of the invention can be used to detect and type degraded RNA sources comprising RNA with a RIN value of less than 8, the probes and primers of the invention can also be used to detect and type RNA sources with a RIN value of 8-10. That is, the primers and probes of the present invention also enable detection and typing of RNA regardless of RIN values.
In one embodiment, the method of the invention functions or enables RNA marker detection when the RNA Integrity (RIN) is less than RIN8, more preferably less than RIN 7, more preferably less than RIN 6, more preferably less than RIN 5, more preferably less than RIN 4, more preferably less than RIN 3, more preferably less than
In particular, the inventors have developed a set of primers specific for the region of 19 markers HBD, SLC4a1, GYPA, FDCSP, HTN3, STATH, PRM1, TGM4, TNP1, PRM2, KLK2, MSMB, MMP10, STC1, MMP3, MMP11, cyp2b7p.l.gass or l.crisp specific for circulating blood, saliva, sperm, semen, menstrual fluid and vaginal material, which allows the identification of samples that may undergo a degree of RNA degradation. Table 1 summarizes the corresponding primers.
RNA detection method
It will be appreciated that any suitable method of detecting RNA may be used in the present invention. Many methods are known in the art and can be used to identify the source of a biological sample.
A wide variety of RNA detection methods are currently available, ranging from non-amplification methods (in situ hybridization, microarray and nanostring counter) to amplification (PCR) -based methods (reverse transcriptase PCR (RT-PCR) and quantitative reverse transcriptase PCR (qRT-PCR)), next generation sequencing (massively parallel sequencing/high throughput sequencing) and RNA aptamers.
In situ hybridization
In Situ Hybridization (ISH) is a type of hybridization that uses labeled complementary DNA or RNA strands (i.e., probes) to locate a particular DNA or RNA sequence in a portion or portion of a tissue (in situ), or in whole tissue (whole mount ISH), in a cell if the tissue is small enough (e.g., plant seeds, drosophila embryos), and in Circulating Tumor Cells (CTCs). This is in contrast to immunohistochemistry, which typically locates proteins in tissue sections.
In situ hybridization is a powerful technique for identifying specific mRNA species within individual cells in tissue sections, providing insight into the physiological processes and pathogenesis of the disease. However, in situ hybridization requires many steps to be taken to precisely optimize each tissue examined and each probe used. In order to maintain the target mRNA in the tissue, it is often necessary to use a cross-linking fixative (e.g., formaldehyde).
Degradation of target RNA is a problem in ISH experiments. The methods of the present invention provide a solution to this problem by targeting a stable region within the target RNA of interest.
Microarray
A DNA microarray (also commonly referred to as a DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to simultaneously measure the expression levels of a large number of genes, or to genotype multiple regions of the genome. Each DNA spot contained picomoles (10)-12Moles) of a specific DNA sequence, referred to as a probe (or reporter molecule or oligonucleotide). These may be short portions of genes or other DNA elements that are used to hybridize under high stringency conditions to cDNA or cRNA (also known as antisense RNA) samples (known as targets). Probe-target hybridization is typically detected and quantified by detecting fluorophore, silver, or chemiluminescent labeled targets to determine the relative abundance of nucleic acid sequences in the target.
The invention is useful for microarray analysis of tissues, including tissues subject to degradation. By designing probes that contain stable regions of targeted RNA (according to the invention) on a microarray chip, microarray analysis can provide a more realistic representation of in vivo expression profiles that are not biased by degradation after RNA extraction from tissue samples. Such chips can also be used to screen samples containing RNA, including degraded RNA, to type the source of the RNA as previously described.
NanoString nCounter
The nCounter technique of NanoString, a variant of DNA microarrays, was invented and patented by Krassen Dimitrov and Dwayne Dunaway. It uses molecular "barcodes" and microscopic imaging to detect and count up to hundreds of unique RNAs in a single hybridization reaction. Each color-coded barcode is linked to a single target-specific probe corresponding to the gene of interest.
The NanoString protocol comprises the following steps:
● hybridization: the NanoString technique uses two to 50 base probes per mRNA hybridized in solution. The reporter probe carries a signal, while the capture probe allows for immobilization of the complex for data collection.
● purification and immobilization: after hybridization, excess probe is removed, and probe/target complexes are aligned and immobilized in an nCounter cassette.
● data collection: the sample cartridge was placed in a digital analysis instrument for data collection. For each target molecule, the color codes on the cassette surface are counted and tabulated.
nCounter analysis system: the system consists of two instruments: prep Station, which is an automated fluidic instrument that immobilizes the CodeSet complex for data collection, and Digital Analyzer, which acquires data by counting fluorescent barcodes. Since the NanoString nCounter system relies on probe-target hybridization for RNA detection and analysis, the present invention is immediately applicable to NanoString nCounter. The NanoString nCounter probe design (target hybridization site) is intended to be designed to meet certain thermodynamic requirements and does not take into account target RNA degradation or stability. Therefore, we believe that by the present invention, nanostring counter RNA detection can be greatly improved by designing probes to hybridize to a stable region in the RNA sequence.
Sample (I)
The sample may be any type of biological sample that contains RNA.
Samples suitable for in situ hybridization include biological tissue sections.
Preferably, the forensic sample is selected from the group comprising blood, semen (with or without sperm), saliva, vaginal material and menstrual fluid.
RNA extraction
RNA extraction procedures are well known to those skilled in the art. Examples include: acid guanidine thiocyanate-phenol-chloroform RNA extraction [64 ]; magnetic bead-based RNA extraction [65 ]; column-based RNA purification [66, 67 ]; and TRIzol (TRI reagent) RNA extraction [68 ].
RNA sequencing and Stable region identification
RNA sequencing is the sequencing of all the RNA in a sample using sequencing commonly known as Next Generation Sequencing (NGS) (second generation sequencing or massively parallel sequencing; [69-72 ]). Although different sequencing instrument manufacturers employ slightly different sequencing chemistries, RNA sequencing can be achieved using any of these NGS (massively parallel sequencing) techniques [69, 73 ]. There are nuances in RNA sequencing methods due to the availability of many NGS technologies. The following is a general description of how NGS is used for RNA sequencing [70 ]:
● Total RNA is extracted from the sample of interest using commonly used RNA extraction methods. Post-extraction processing can be used to enrich RNA samples.
● complementary DNA (cDNA) is then synthesized using the extracted RNA. The cDNA was then used as a template for RNA sequencing.
● NGS uses variants of Sequencing By Synthesis (SBS) chemistry [74 ]. New nucleotide fragments (called reads) were synthesized base by base using cDNA as a template, and each introduced base was recorded during the sequencing process [74 ].
● the data output for RNA sequencing is a list of all reads generated and their sequences [74, 70 ]. The data was evaluated for quality [75 ]. For RNA sequencing, sequencing reads are then aligned to a reference genome using a splice-aware sequence alignment algorithm [76 ].
The alignment can then be visualized using any genome browser or sequence viewing software. The RNA stability region is identified by observing sequencing read alignments along the RNA of interest. Regions along the RNA sequence with more aligned reads (high read coverage) are considered stable regions.
Stable region
A stabilizing region of an RNA sequence according to the invention is a region within any given RNA sequence for which RNA sequencing data shows that it produces more aligned sequencing reads than at least one other region of the same RNA sequence.
PCR-based methods
PCR-based methods are particularly preferred for detecting RNA sequences in the methods of the invention.
General PCR methods are well known to those skilled in the art [77 ]. Various other developments of the basic PCR method can also be advantageously applied in the method of the invention. Examples are briefly discussed below.
Multiplex PCR
Multiplex PCR utilizes multiple primer sets in a single PCR reaction to produce amplification products (amplicons) of different sizes that are specific for different target RNA, cDNA, or DNA sequences. By targeting multiple sequences at once, diagnostic information can be obtained from a single reaction that would otherwise require several times the reagents and more time to perform. The annealing temperature and primer set are typically optimized to work in a single reaction and produce different amplicon sizes. That is, the amplicons should form distinct bands when viewed by gel electrophoresis or capillary electrophoresis. Multiplex PCR can be used in the methods of the invention to distinguish the sample types that are used in a single sample or reaction.
MLPA
Multiplex ligation-dependent probe amplification (MLPA) (US 6,955,901) is a variant of the multiplex polymerase chain reaction that allows amplification of multiple targets with only a single primer pair. Each probe consists of two oligonucleotides that recognize adjacent target sites on DNA. One probe oligonucleotide contains a sequence recognized by the forward primer and the other probe oligonucleotide contains a sequence recognized by the reverse primer. Only when these two probe oligonucleotides hybridize to their respective targets, they can be ligated into a complete probe. The advantage of splitting the probe into two parts is that only the ligated oligonucleotide is amplified, and not the unbound probe oligonucleotide. If the probes are not separated in this manner, primer sequences at either end will allow the probes to be amplified regardless of whether they hybridize to the template DNA. Each intact probe has a unique length so that the amplicons it produces can be isolated and characterized (e.g., by capillary electrophoresis and other methods). Since the forward primer used for probe amplification is fluorescently labeled, each amplicon produces a fluorescence peak that can be detected by a capillary sequencer. The presence or absence (or relative amount) of each amplicon can be determined by comparing the peak pattern obtained on a given sample with the peak pattern measurements obtained on a variety of reference samples. This then indicates the presence or absence (or relative amount) of the target sequence present in the sample DNA. Products can also be detected using gel electrophoresis or microfluidic systems such as shimadzu multina. The reference sample is used to determine if the presence is the same. More information about MLPA can be found in web http: // www.mlpa.com. MLPA probes can be synthesized as oligonucleotides by methods known to those skilled in the art. MLPA probes and reagents are commercially produced and purchased from HRC-Holland (http:// www.mlpa.com).
Quantitative PCR
Quantitative PCR (Q-PCR) is used to measure the amount of PCR product (usually in real time). Q-PCR quantitatively measures the initial amount of DNA, cDNA or RNA. Q-PCR is commonly used to determine the presence and copy number of DNA sequences in a sample. Quantitative real-time PCR has a very high accuracy. The Q-PCR method uses a fluorescent dye (e.g., SYBR Green, EvaGreen) or a fluorophore-containing DNA probe (e.g., TaqMan) to measure the amount of an amplification product in real time. Q-PCR is sometimes abbreviated RT-PCR (real-time PCR) or RQ-PCR, QRT-PCR or RTQ-PCR.
Primer and method for producing the same
The term "primer" refers to a short polynucleotide, typically having a free 3' OH group, that hybridizes to a template and is used to initiate polymerization of a polynucleotide complementary to the template. Such primers preferably have a length of at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides.
In conventional primer design for amplification of RNA marker sequences, primers are typically designed to cover exon boundaries to prevent amplification of genomic DNA.
The present invention relates to a stable region of a targeted RNA transcript that is particularly useful in amplifying a marker from a degraded sample. It is evident that once a stable region is identified, this region can be used to type samples containing RNA with RIN values between 8 and 10 and below 8. Thus, these two options form part of the present invention.
In one embodiment, the primers of the invention used in the methods of the invention do not cross exon boundaries.
Although not preferred, in one embodiment, the primers of the invention used in the methods of the invention may span exon boundaries.
Labeling of primers
Methods for labeling primers are well known to those skilled in the art and include:
the primers may be labeled enzymatically [78] or chemically [79] including automated solid phase chemical synthesis ].
The primers can be labeled with: fluorescent markers (fluorophores, [80]), biotin [81], or radioactive and nonradioactive markers (e.g., digoxigenin) [82 ].
Primers labeled by these methods form part of the invention.
Probe-based method
Probe-based methods can be employed in the methods of the invention to detect RNA sequences. Methods for hybridizing probes to target nucleic acid sequences are well known to those skilled in the art [83 ].
Probe-based methods include in situ hybridization.
The term "probe" refers to a short polynucleotide used to detect a polynucleotide sequence that is at least partially complementary to the probe in a hybridization-based assay. A probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably, such probes are at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length.
Labeling of probes
Methods for labeling probes are well known to those skilled in the art and include:
the probes may be labeled enzymatically [83, 78] or chemically [79], including automated solid phase chemical synthesis.
The probe may be:
molecular beacons [84], TaqMan [80], Scorpion [85], in situ hybridization probes [86], radioactive and nonradioactive [87, 82 ].
Probes labeled by these methods form part of the present invention.
Polynucleotide
As used herein, the term "polynucleotide" refers to a single-or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, but preferably at least 5 nucleotides, and includes the following as non-limiting examples: coding and non-coding sequences of the gene, sense and antisense sequence complements, exons, introns, genomic DNA, cDNA, pre-mRNA, rRNA, siRNA, miRNA, tRNA, naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, and fragments thereof. In one embodiment, the nucleic acid is isolated from its normal cellular environment. The term "nucleic acid" is used interchangeably with "polynucleotide".
Method for extracting nucleic acid
Methods for extracting nucleic acids are well known to those skilled in the art [83 ].
As discussed in the examples section, specialized extraction procedures may optionally be applied depending on the sample type. For example, RNA from forensic samples can be extracted using DNA-RNA co-extraction methods, as described by Bowden et al 2011 [88 ].
All such methods are intended to be included within the scope of the present invention.
Percent identity
Variant polynucleotide sequences preferably exhibit at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99%. Identity is determined over a comparison window of at least 10 nucleotide positions, more preferably at least 11 nucleotide positions, more preferably at least 12 nucleotide positions, more preferably at least 13 nucleotide positions, more preferably at least 14 nucleotide positions, more preferably at least 15 nucleotide positions, more preferably at least 16 nucleotide positions, more preferably at least 17 nucleotide positions, more preferably at least 18 nucleotide positions, more preferably at least 19 nucleotide positions, more preferably at least 20 nucleotide positions, more preferably at least 21 nucleotide positions and most preferably the entire length of a given polynucleotide sequence. The present invention includes these variations.
Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequences were compared to candidate polynucleotide sequences using BLASTN (from the BLAST suite of programs, version 2.2.5[2002, 11 months ]) in bl2seq [89], publicly available from NCBI (ftp:// ftp. NCBI. nih. gov/BLAST /). The default parameters for bl2seq are used, except that the filtering of the low complexity part should be turned off.
The polynucleotide sequences can be checked for identity using the following unix command line parameters:
bl2seq-i nucleotideseq1-j nucleotideseq2-F-p blastn
parameter-F turns off the filtering of the low complexity portion. The parameter-p selects the appropriate algorithm for the sequence pair. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in the row "Identities ═ a".
Polynucleotide sequence identity may also be calculated over the entire length of the overlap between the candidate and subject polynucleotide sequences using a global sequence alignment program (e.g., Needleman-Wunsch; [90 ]). In the case of a http: a complete implementation of the Needleman-Wunsch global alignment algorithm was found in the needle program of// www.hgmp.mrc.ac.uk/Software/EMBOSS package [91] obtained. The european bioinformatics institute server is also in http: the/www.ebi.ac.uk/EMBOSS/align/in-line provides a tool to perform EMBOSS-needle global alignment between two sequences.
Alternatively, sequence identity can be calculated using the GAP program, which calculates the optimal global alignment of two sequences without penalty for end GAPs (penalizing) [92 ].
Sequence identity can also be calculated by aligning the sequences to be compared using Vector NTI version 9.0, using the Clustal W algorithm [93 ]]Then Vector NTI version 9.0 (9/2/2003)1994-2003 InforMax, to Invitrogen) to calculate the percent sequence identity between the aligned sequences.
Thus, in general, the invention provides a method for detecting an RNA sequence in a sample. The method comprises the following steps:
a) providing a sample, and
b) the RNA sequence is detected using at least one primer or probe complementary to a stabilizing region of the RNA sequence.
The stable region of the RNA sequence is preferably identified using RNA sequencing of the sample, and in particular, identified as a region of the RNA sequence that has more aligned sequencing reads than another region or regions of the same RNA sequence.
The stabilizing region has been identified and discussed herein, and the stabilizing region used in the methods of the invention may be selected from the group consisting of SEQ ID NOs: 1 to SEQ ID NO: 19 or the complement of either thereof.
Primers are also identified and discussed herein, and may be selected from the group consisting of SEQ ID NOs: 20 to SEQ ID NO: 57 or the complement of either thereof.
In addition, in more specific cases, it can be seen that the present invention encompasses polypeptides that are homologous to a nucleotide sequence selected from SEQ ID NOs: 1 to SEQ ID NO: 19 or the complement thereof, a nucleotide sequence comprising at least 5 nucleotides having at least 70% identity to the sequence of seq id no.
Furthermore, and again in a more specific context, it can be seen that the present invention includes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to SEQ ID NO: 19 or the complement thereof, or a sequence of at least 5 nucleotides of the sequence.
Furthermore, and again in a more specific context, it can be seen that the present invention encompasses polypeptides that are homologous to a nucleotide sequence selected from SEQ ID NOs: 1 to SEQ ID NO: 19 or the complement thereof, a nucleotide sequence comprising at least 10 nucleotides having at least 70% identity to the sequence of seq id no.
Furthermore, and again in a more specific context, it can be seen that the present invention includes a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1 to SEQ ID NO: 19 or its complement, or a sequence of at least 10 nucleotides of the sequence.
Furthermore, and again in a more specific case, it can be seen that the invention comprises a polypeptide selected from the group consisting of SEQ ID NOs: 20 to SEQ ID NO: 57.
The use of a nucleotide sequence as defined above for typing of a sample comprising RNA forms in particular part of the present invention.
It is apparent that samples comprising RNA can be obtained from a variety of sources. The most preferred sample is a biological tissue sample, which may be a solid or a liquid.
The method of the invention is particularly suitable for use in the field of forensic medicine, and the sample may thus be any type of forensic sample comprising RNA, e.g. selected from the group comprising blood, semen (with or without sperm), saliva, vaginal material and menstrual fluid.
Preferably, the RNA is extracted from the sample prior to the detection step and the RNA sequence may be detected directly or indirectly as known to those skilled in the art. However, it is preferred to detect the RNA sequence indirectly by detecting complementary DNA (cDNA) corresponding to the RNA sequence.
In more specific cases, it can also be seen that the present invention comprises a method for typing a sample comprising RNA, wherein said method comprises the steps of:
a) providing a sample comprising RNA;
b) detecting one or more RNA sequences in the sample using at least one primer or probe complementary to one or more stabilizing regions of the RNA;
wherein the stabilized RNA sequence is specific for a sample type; and is
Wherein detection of the stabilized RNA sequence indicates the type of sample.
In another aspect, it can be seen that the present invention includes a method of typing a sample comprising degraded RNA, the method comprising the steps of:
a) providing a sample comprising degraded RNA;
b) detecting one or more stable RNA sequences in the sample using at least one primer or probe complementary to one or more stable regions of the degraded RNA;
wherein the stabilized RNA sequence is specific for the type of sample; and is
Wherein detection of the target RNA sequence indicates the type of sample.
In another embodiment, the invention can be a method for identifying a stable region of RNA in a sample, the method comprising:
a) providing a sample comprising RNA, and providing a sample,
b) isolating the total RNA from the sample,
c) the DNA is removed from the sample and,
d) generating cDNA complementary to the RNA in the sample,
e) the cDNA was sequenced.
Wherein a stable region of an RNA sequence is identified as a region of the RNA sequence that has more aligned sequencing reads than another region or regions of the same RNA sequence.
As previously mentioned, this method can be applied to RNA that has degraded to conditions previously thought to be unusable as a means of typing/identifying the source of the sample from which the RNA was extracted. The methods of the invention can be used to type/identify the source of a sample in which the RNA content has a RIN value of less than 8. Since stable regions in RNAs with values less than 8 will also be present in RNAs with RIN values of 8 to 10, once stable regions are identified, those stable regions can also be used to identify/type the source of the sample with RIN values of 8 to 10. Thus, the method can be used to type/identify the source of samples having any RIN value, including samples for which RIN values cannot be determined.
As previously described, a stable region of an RNA sequence can be identified as a region of the RNA sequence that has more aligned sequencing reads than another region or regions of the same RNA sequence.
It will be apparent to the skilled person that it is preferred to use primers or probes for detecting RNA sequences. It is also apparent that more than one primer or probe (e.g., two primers) can be used to detect RNA sequences, if appropriate/desired.
The primers and/or probes should preferably correspond to, or be complementary to, or be capable of hybridizing to, sequences within the stable region of RNA extracted from the sample. The primers are used to amplify a portion of the stability region to which the primers bind, for example, by Polymerase Chain Reaction (PCR) methods. The PCR method may be selected from standard PCR, reverse transcriptase PCR (RT-PCR) and quantitative reverse transcriptase PCR (qRT-PCR).
In addition, and as will also be apparent to the skilled person, probes may be used to detect RNA sequences. This preferably corresponds to, or is complementary to, a sequence within the RNA stabilizing region extracted from the sample.
The RNA sequence may be encoded by a marker gene specific for the type of sample. That is, expression of an RNA sequence or presence of an RNA sequence in a sample is diagnostic of the type of sample. For example, when the sample is circulating blood, the marker gene is selected from the group consisting of:
● Hemoglobin (HBD), and/or
● solute Carrier family 4 (anion exchange protein), Member 1(Diego blood group) (SLC4A1),
● glycophorin A (GYPA).
When the sample comprises saliva, the marker gene is selected from:
● Follicular Dendritic Cell Secretory Protein (FDCSP), and/or
● Histone 3(HTN3)
● Sialoprotein (STATH).
When the sample comprises sperm, the marker gene is selected from the group consisting of:
● protamine 1(PRM1), and/or
● transition protein 1 (during replacement of histone by protamine) (TNP1) and/or
● protamine 2(PRM 2).
When the sample is semen, the marker gene is selected from the group consisting of:
● kallikrein-related peptidase 2(KLK2), and/or
● microplasmin beta (MSMB) and/or
● Transglutaminase 4(TGM 4).
When the sample is menstrual fluid, the marker gene is selected from the group consisting of:
● matrix metallopeptidase 10(MMP10), and/or
● Stannyzing calmodulin 1(STC1), and/or
● matrix metallopeptidase 3(MMP3)
● matrix metallopeptidase 11(MMP 11).
When the sample is vaginal material, the marker gene is selected from the group consisting of:
●
● Lactobacillus crispatus protein (L.gass) and/or
● Lactobacillus gasseri protein (L.crisp).
The detection method of the present invention may comprise the use of a primer or probe capable of hybridizing to a stable region of an RNA sequence or a cDNA corresponding to a stable region or its complement. The method may comprise typing the sample using only one pair of primers or a single probe. Alternatively, multiple pairs of primers or multiple probes may be used.
The primer or probe may comprise (i) a sequence that is identical to SEQ ID NO: 1 to 19 or any portion of its complement, or (ii) a sequence of at least 5 nucleotides having at least 70% identity to any portion of the sequence of any one of SEQ ID NOs: 1 to 19 or a sequence of at least 5 nucleotides having at least 70% identity to a sequence of any one of SEQ ID NOs: 1 to 19 or a sequence of at least 5 nucleotides of a sequence of any one of SEQ ID NOs: 1 to 19 or the complement thereof, or (v) a sequence of at least 5 nucleotides selected from the sequences of SEQ ID NOs: 20 to 57, or (vi) a tag or label linked to a sequence selected from any of these sequences.
The primer or probe may comprise (i) a sequence that is identical to SEQ ID NO: 1 to 19 or any portion of its complement having at least 70% identity to a sequence of at least 10 nucleotides, or (ii) a sequence of at least 10 nucleotides having at least 70% identity to any portion of the sequence of any one of SEQ ID NOs: 1 to 19 or a sequence of at least 10 nucleotides having at least 70% identity to a sequence of any one of SEQ ID NOs: 1 to 19 or a sequence of at least 10 nucleotides of a sequence of any one of SEQ ID NOs: 1 to 19 or a sequence of at least 10 nucleotides of a sequence of any one of SEQ ID NOs: 20 to 57, or (vi) a tag or label linked to a sequence selected from any of these sequences.
For example, typing of a sample can be performed using multiplex PCR with multiple primers, wherein at least one primer diagnoses the type of sample.
Preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20, more preferably at least 21, more preferably at least 22, more preferably at least 23, more preferably at least 24, more preferably at least 25, more preferably at least 26, more preferably at least 27, more preferably at least 28, more preferably at least 29, more preferably at least 30, more preferably at least 31, more preferably at least 32, more preferably at least 33, more preferably at least 34, more preferably at least 35, carbon dioxide is used, More preferably at least 36, more preferably at least 37, more preferably at least 38 primers of the invention are used for multiplex PCR.
The invention also allows to provide a kit comprising at least one primer or probe according to the invention. Such a kit may comprise any number of primers or probes, in particular a kit may comprise at least 2, more preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20, more preferably at least 21, more preferably at least 22, more preferably at least 23, more preferably at least 24, more preferably at least 25, more preferably at least 26, more preferably at least 27, more preferably at least 28, more preferably at least 29, more preferably at least 30, more preferably at least 31, more preferably at least 32, more preferably at least 33, more preferably at least 34, more preferably at least 35, more preferably at least 36, more preferably at least 37, more preferably at least 38 primers or probes of the invention. Combinations of primers and probes may also be provided in such kits.
It will be apparent that the kit will also contain instructions for use, if such instructions are required.
The invention also allows for the provision of a microarray or chip or similar product comprising a sequence of a stable region of RNA or a complementary sequence thereof which has been identified herein as useful for typing/identification of a sample. These sequences have been used to generate primers and probes useful in microarrays or chips or similar products for detecting nucleotide sequences.
Such microarrays or chips are of particular commercial importance because they enable efficient and accurate identification of unknown samples containing RNA, including where the RNA has degraded. Once these products have the benefit of the knowledge of the present invention, they can be produced within the capabilities of those skilled in the art.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：来源于根瘤菌种细菌的纳米囊泡及其用途