阅读说明：本技术 一种检测高度片段化样本的变性增强数字微滴pcr方法 (Denaturation-enhanced digital droplet PCR method for detecting highly fragmented samples ) 是由 陈谦 刘弘扬 刘泓 李新新 贾海雪 于 2019-12-03 设计创作，主要内容包括：本发明公开了一种检测高度片段化样本的变性增强数字微滴PCR方法。本发明有助于提高痕量高度片段化游离DNA检测灵敏度和特异性。并提供了几组检测游离DNA的寡核苷酸的序列。(The invention discloses a denaturation-enhanced digital microdroplet PCR method for detecting a highly fragmented sample. The invention is beneficial to improving the sensitivity and specificity of the detection of trace high-fragmentation free DNA. And provides several sets of oligonucleotide sequences for detecting free DNA.)

1. A denaturing-enhanced digital droplet PCR method of detecting highly fragmented samples, comprising the steps of:

(1) extracting free DNA from the sample, extracting DNA from the sample,

(2) and (3) carrying out end repair on the extracted free DNA, wherein the reaction conditions of the end repair are as follows: keeping the temperature at 30 ℃ for 10-20 minutes; keeping the temperature at 65 ℃ for 10-20 minutes; obtaining end repair free DNA;

(3) using the end repair free DNA as a template, carrying out microdroplet generation by using a Raindrop digital PCR System, and then carrying out PCR reaction, wherein the PCR reaction procedure is as follows: 10 minutes at 95 ℃;

repeat 50 cycles: 30 seconds at 94 ℃ and 1 minute at 56 ℃;

10 minutes at 98 ℃ and finally 10 ℃ to terminate;

(4) after the PCR reaction is finished, the Raindrop digital PCR System is used for reading the experimental result, and the copy number of the fluorescent liquid drop carrying the corresponding fluorescent liquid is recorded.

2. The method of denatured-enhanced digital microdroplet PCR of claim 1, wherein the sample is selected from the group consisting of plasma DNA, urine DNA, sweat DNA, saliva DNA, semen DNA, pleural fluid DNA, ascites DNA, stool DNA, fossil DNA, paraffin-embedded DNA.

3. The denaturing-enhanced digital droplet PCR method of claim 2, wherein in step (2) the end-repair reaction system is: 10ng of free DNA, 2. mu.L of end-repairing enzyme, 5. mu.L of reaction buffer, Tris-HCl,10mM, and a volume of 50. mu.L of reaction system.

4. The denaturing-enhanced digital droplet PCR method of claim 1, wherein in step (4), the species that detect fluorescence comprise FAM, HEX, VIC.

5. A denaturing-enhanced digital droplet PCR method of detecting highly fragmented samples, comprising the steps of:

(A) extracting free DNA from the sample, extracting DNA from the sample,

(B) the extracted free DNA was subjected to single tube denaturation-enhanced digital PCR: adding water into the extracted free DNA X10 ng, 20X end repair enzyme 0.5 muL, 2X ddPCR reaction buffer solution 10 muL, upstream and downstream primers 1 muL respectively, Taqman probe-10.5 muL, Taqman probe-20.5 muL, and fixing the volume to 20 muL,

droplet generation was performed using a Raindrop digital PCR System followed by PCR reactions with the following protocol: 10-20 minutes at 30 ℃, 10-20 minutes at 65 ℃,

repeat 50 cycles: 10 minutes at 95 ℃, 30 seconds at 94 ℃ and 1 minute at 56 ℃;

10 minutes at 98 ℃ and finally 10 ℃ to terminate;

(C) after the PCR reaction is finished, the Raindrop digital PCR System is used for reading the experimental result, and the copy number of the fluorescent liquid drop carrying the corresponding fluorescent liquid is recorded.

6. The method of denatured-enhanced digital microdroplet PCR of claim 1, wherein the sample is selected from the group consisting of plasma DNA, urine DNA, sweat DNA, saliva DNA, semen DNA, pleural fluid DNA, ascites DNA, stool DNA, fossil DNA, paraffin-embedded DNA.

7. The denaturing-enhanced digital droplet PCR method of claim 1, wherein in step (C), the species that detect fluorescence comprise FAM, HEX, VIC.

8. A group of oligonucleotides for detecting free DNA is characterized by consisting of oligonucleotides with base sequences shown in sequence tables SEQ ID No.1 to SEQ ID No. 4; wherein SEQ ID No.1 and SEQ ID No.2 are respectively an upstream primer and a downstream primer for amplifying the BRAF gene, and SEQ ID No.3 and SEQ ID No.4 are respectively probes for amplifying the V600E locus of the BRAF gene.

9. A group of oligonucleotides for detecting free DNA, which is characterized by consisting of oligonucleotides with base sequences shown in sequence tables SEQ ID No.5 to SEQ ID No. 7; wherein SEQ ID No.5 and SEQ ID No.6 are respectively an upstream primer and a downstream primer for amplifying the BRAF gene, and SEQ ID No.3 and SEQ ID No.7 are respectively probes for amplifying the V600K locus of the BRAF gene.

10. A group of oligonucleotides for detecting free DNA, which is characterized by consisting of oligonucleotides with base sequences shown in sequence tables SEQ ID No.8 to SEQ ID No. 11; wherein SEQ ID No.8 and SEQ ID No.9 are upstream and downstream primers for amplifying NRAS gene respectively, and SEQ ID No.10 and SEQ ID No.11 are probes for amplifying Q61K locus of NRAS gene respectively.

Technical Field

The invention relates to the technical field of biology, in particular to a denaturation-enhanced digital microdroplet PCR method for detecting a highly fragmented sample.

Background

The examination of trace clinical samples, such as fluid biopsies of ctDNA, is of great interest because of its potential as a clinically important biomarker. At present, accurate medicine faces a difficult challenge in the following aspects: 1) the samples are few: 2-3ml pregnant woman peripheral blood has only 1 fetal cell; 1ml of whole blood has only 1-100 circulating tumor cells; however, the peripheral blood (free DNA) of pregnant women and tumor patients contains much less cfDNA and ctDNA. 2) Strong interference: the heterogeneity of the tumor and the large number of normal tissue cells surrounding the tumor lead to tens of thousands of times of other interferences around the target nucleic acid fragments to be detected. 3) The conventional PCR reaction system requires at least 10 molecules, has an error of 5% or more, and is difficult to detect 1 mutant molecule among 3 ten thousand wild molecules. 4) Conventional PCR cannot resolve differences in gene expression (or copy number differences) by a factor of 2 or less, and it is difficult to identify alleles with frequencies below 1%. Compared with the traditional PCR (qPCR) technology, the digital PCR technology has great advantages in many aspects and is expected to become a gold standard technology for quantitative diagnosis of nucleic acid in the future: 1) absolute quantification without a standard curve; 2) the sensitivity is ultrahigh and reaches 1/million; 3) high precision; 4) directly reaching the lowest detection limit of the single copy level; 5) high tolerance, interference resistance, etc.

When researchers perform biological research, it is necessary to extract nucleic acids (DNA, or RNA) from various types of samples. In which a body fluid sample contains highly fragmented DNA, also called free DNA, and biological detection of DNA in the sample is an important detection step. For example, plasma DNA is extracellular DNA in blood and exists in the form of nucleosomes (DNA-protein complexes). Such fragmented DNA is several tens to several hundreds of base pairs long (166 bp as the main peak). Such fragmented DNA is usually released into the blood circulation system from a small number of apoptotic cells, and the content is extremely low, and it is difficult to detect reliable information by the existing PCR method.

Despite some advances, digital PCR techniques are limited by the limited number of DNA copies available for analysis. When there is only a nanogram-level ctDNA, the information obtained can be affected by statistical sampling errors and the number of clinically relevant targets available for analysis can be reduced. In liquid biopsy, starting material deficiency is a bottleneck for digital PCR and liquid biopsy, because the detection limit of digital PCR technology cannot exceed the upper DNA input limit regardless of analytical sensitivity.

The nested PCR has the effect of enriching trace DNA, but the nested PCR is easy to bring non-specific amplification, and the detection accuracy is influenced. In short amplification fragment PCR, the size of an amplicon fragment is small, so that the design range of a primer is small, and the specificity is difficult to ensure.

Disclosure of Invention

The invention aims to provide a denaturation-enhanced digital microdroplet PCR method for detecting a highly fragmented sample, which improves the detection sensitivity of highly fragmented free DNA, is easy to standardize, has small batch difference, is less in non-specific amplification compared with nested PCR, and has strong specificity compared with short-fragment PCR.

In order to solve the above technical problems, the present invention provides a denaturation-enhanced digital droplet PCR method for detecting a highly fragmented sample, comprising the following steps:

(1) extracting cfDNA from the sample,

(2) performing end repair on the extracted cfDNA, wherein the reaction conditions of the end repair are as follows: keeping the temperature at 30 ℃ for 10-20 minutes; keeping the temperature at 65 ℃ for 10-20 minutes; obtaining end repair free DNA;

(3) using the obtained end repair free DNA as a template, carrying out microdroplet generation by using a Raindrop digital PCR System, and then carrying out PCR reaction, wherein the PCR reaction procedure is as follows: 8-10 minutes at 95 ℃;

repeat 50 cycles: 20-35 seconds at 94 ℃ and 1-3 minutes at 56 ℃;

10-15 minutes at 98 ℃ and finally 10 ℃ is stopped;

(4) after the PCR reaction is finished, the Raindrop digital PCR System is used for reading the experimental result, and the copy number of the fluorescent liquid drop carrying the corresponding fluorescent liquid is recorded.

In a preferred embodiment of the present invention, the sample is selected from the group consisting of plasma DNA, urine DNA, sweat DNA, saliva DNA, semen DNA, pleural fluid DNA, ascites DNA, stool DNA, fossil DNA, and paraffin-embedded DNA.

In the preferred technical scheme of the invention, in the step (2), the end repairing reaction system is as follows: 10ng of free DNA, 2. mu.L of end-repairing enzyme, 5. mu.L of reaction buffer, and 50. mu.L of Tris-HCl (10mM) was added to the reaction system.

In the preferred technical scheme of the invention, in the step (3), the reaction system is as follows: 2 mu L of free DNA with repaired tail end, 10 mu L of 2XddPCR reaction buffer solution, 1 mu L of upstream primer, 1 mu L of downstream primer, 10.5 mu L of Taqman probe and 20.5 mu L of Taqman probe, and adding water to a constant volume reaction system to 20 mu L.

In a preferred embodiment of the present invention, in step (4), the types of fluorescence to be detected include FAM, HEX, and VIC.

The technical noun is as follows: cfDNA refers to degraded DNA fragments released into plasma, either circulating free DNA or cell free DNA.

The invention provides a denaturation-enhanced digital droplet PCR method for detecting a highly fragmented sample, which comprises the following steps:

(A) extracting cfDNA from the sample,

(B) the extracted free DNA was subjected to single tube denaturation-enhanced digital PCR: adding 10ng of the extracted cfDNA, 0.5 mu L of 20X end repair enzyme, 10 mu L of 2X ddPCR reaction buffer solution, 1 mu L of each of upstream and downstream primers, 10.5 mu L of Taqman probe and 20.5 mu L of Taqman probe into water to fix the volume to 20 mu L,

droplet generation was performed using a Raindrop digital PCR System followed by PCR reactions with the following protocol: 10-20 minutes at 30 ℃, 10-20 minutes at 65 ℃,

repeat 50 cycles: 10 minutes at 95 ℃, 30 seconds at 94 ℃ and 1 minute at 56 ℃;

10 minutes at 98 ℃ and finally 10 ℃ to terminate;

(C) after the PCR reaction is finished, the Raindrop digital PCR System is used for reading the experimental result, and the copy number of the fluorescent liquid drop carrying the corresponding fluorescent liquid is recorded.

In a preferred embodiment of the present invention, the sample is selected from the group consisting of plasma DNA, urine DNA, sweat DNA, saliva DNA, semen DNA, pleural fluid DNA, ascites DNA, stool DNA, fossil DNA, and paraffin-embedded DNA.

In a preferred embodiment of the present invention, in step (C), the types of fluorescence to be detected include FAM, HEX, and VIC.

The third aspect of the present invention provides a set of oligonucleotides for detecting free DNA, which can be used in the primer composition of the above detection method, consisting of oligonucleotides having base sequences represented by SEQ ID No.1 to SEQ ID No.4 of the sequence Listing; wherein, SEQ ID No.1 and SEQ ID No.2 are respectively an upstream primer and a downstream primer for amplifying the BRAF gene, and SEQ ID No.3 and SEQ ID No.4 are respectively probes for amplifying the V600E locus of the BRAF gene.

BRAF-15-F2	AGACCTCACAGTAAAAATAGGT	SEQ ID No.1
			BRAF-15-R2	ACAACTGTTCAAACTGATGG	SEQ ID No.2
BRAF-WT	6FAM-TCTAGCTACAGTGAAATCTCGA-BHQ1a	SEQ ID No.3
			BRAF-V600E-Mut	HEX-TCTAGCTACAGAGAAATCTCGA-BHQ1	SEQ ID No.4

The invention also provides a group of oligonucleotides for detecting free DNA, which can be used for the primer composition of the detection method and consists of oligonucleotides with base sequences shown in sequence tables SEQ ID No.5 to SEQ ID No. 7; wherein SEQ ID No.5 and SEQ ID No.6 are respectively an upstream primer and a downstream primer for amplifying the BRAF gene, and SEQ ID No.3 and SEQ ID No.7 are respectively probes for amplifying the V600K locus of the BRAF gene.

BRAF1–15-F1	TTTCTTCATGAAGACCTCACA	SEQ ID No.5
			BRAF2-R1	CCACAAAATGGATCCAGACAACTGT	SEQ ID No.6
BRAF-WT	6FAM-TCTAGCTACAGTGAAATCTCGA-BHQ1	SEQ ID No.3
			BRAF-V600K-Mut	HEX-TCTAGCTACAAAGAAATCTCGAT-BHQ1	SEQ ID No.7

The invention also provides a group of oligonucleotides for detecting free DNA, which can be used for the primer composition of the detection method and consists of oligonucleotides with base sequences shown in sequence tables SEQ ID No.8 to SEQ ID No. 11; wherein SEQ ID No.8 and SEQ ID No.9 are upstream and downstream primers for amplifying NRAS gene respectively, and SEQ ID No.10 and SEQ ID No.11 are probes for amplifying Q61K locus of NRAS gene respectively.

NRAS-3-F2	GTGAAACCTGTTTGTTGGA	SEQ ID No.8
			NRAS-3-R2	GTCCTCATGTATTGGTCTCT	SEQ ID No.9
NRAS-WT	6FAM-ACAGCTGGACAAGAAGAGTACA-BHQ1	SEQ ID No.10
			NRAS-Q61K-Mut	HEX-ACAGCTGGAAAAGAAGAGTACA-BHQ1	SEQ ID No.11

The invention also provides a group of oligonucleotides for detecting free DNA, which can be used for the primer composition of the detection method and consists of oligonucleotides with base sequences shown in sequence tables SEQ ID No.12 to SEQ ID No. 15; SEQ ID No.12 and SEQ ID No.13 are upstream and downstream primers for amplifying EGFR gene, respectively, and SEQ ID No.14 and SEQ ID No.15 are probes for amplifying the L858R site of EGFR gene, respectively.

EGFR p.L858R Forward primer	GCAGCATGTCAAGATCACAGATT	SEQ ID No.12
			EGFR p.L858R Reverse primer	CCTCCTTCTGCATGGTATTCTTTCT	SEQ ID No.13
EGFR p.L858R WT probe	VIC-AGTTTGGCCAGCCCAA-MGB-NFQ	SEQ ID No.14
			EGFR p.L858R MT probe	6FAM-AGTTTGGCCCGCCCAA-MGB-NFQ	SEQ ID No.15

The invention improves the initial template amount by modifying the template DNA into single strands and repairing the single strands so as to achieve the method for improving the sensitivity and the specificity of the trace free DNA detection.

Drawings

The invention is further described with reference to the following figures and examples:

FIG. 1A shows the result of the conventional ddPCR assay in example 1, and FIG. 1B shows the result of the enhanced ddPCR assay in example 1; the capability of enhancing the digital PCR to detect the mutant DNA copy is shown to be stronger than that of the common ddPCR.

FIG. 2 is a statistical chart of comparison of copy number detected by BRAFV600E different species samples and experimental methods, and the capability of detecting mutant DNA copy by enhanced digital PCR is stronger than that of common ddPCR.

FIG. 3 is a statistical chart of copy number comparison of EGFRL858R samples of different species and experimental methods, and the copy capacity of the enhanced digital PCR method for detecting mutant DNA is stronger than that of the ordinary ddPCR method.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. The following describes embodiments of the present invention with reference to the drawings.

Sample(s)

Peripheral blood samples of patients validated to carry BRAFV600E and L858R mutations

Reagent and apparatus

QIAamp DNA FFPE Tissue Kit (Qiagen), genomic DNA extraction Kit AllPrep DNA/RNAmini Kit (Qiagen), dNTP Mix (2.5mM) (Takara,4030), agarose (Tiangen Biochemical, RT101),