阅读说明：本技术 一种用于血流感染病原诊断的血浆游离dna标志物 (Plasma free DNA marker for diagnosis of blood stream infection pathogen ) 是由 申奥 王晓凤 吴红龙 于 2020-04-21 设计创作，主要内容包括：本发明公开了一种用于血流感染病原诊断的血浆游离DNA标志物。本发明提供了一种血浆游离DNA的处理方法,包括如下步骤：从待测血浆游离DNA中选择大小为60-140bp的片段,该方法可作为通过血浆游离DNA进行血流感染病原体检测的样本前处理方法。本发明对大肠埃希菌、肺炎克雷伯菌、金黄色葡萄球菌、屎肠球菌、鲍曼不动杆菌感染的血浆样本中游离核酸分布范围进行了研究,确定了这5种细菌在血浆中的游离核酸片段分布。本发明对于提高血浆宏基因组检测中病原检测的灵敏度,指导设计PCR扩增产物片段长度范围,以提高PCR检测灵敏度具有重要意义。本发明为血浆游离DNA检测产品开发过程中的病原模拟样本制备提供依据。(The invention discloses a plasma free DNA marker for diagnosing blood stream infection pathogens. The invention provides a method for treating free DNA of blood plasma, which comprises the following steps: the method can be used as a sample pretreatment method for detecting blood stream infection pathogens through the plasma free DNA. The invention researches the distribution range of free nucleic acid in plasma samples infected by Escherichia coli, Klebsiella pneumoniae, staphylococcus aureus, enterococcus faecium and Acinetobacter baumannii, and determines the distribution of the free nucleic acid fragments of the 5 bacteria in the plasma. The invention has important significance for improving the sensitivity of pathogen detection in plasma macro genome detection and guiding the length range of PCR amplification product fragments to be designed so as to improve the sensitivity of PCR detection. The invention provides a basis for preparing the pathogen simulation sample in the development process of the plasma free DNA detection product.)

1. A method for processing free DNA in plasma comprises the following steps: fragments of 60-140bp in size were selected from the plasma free DNA to be tested.

2. A sample pretreatment method for detecting blood stream infection pathogens through plasma free DNA comprises the following steps:

(A1) extracting free DNA from blood plasma to be detected, performing end repair and adding A, and connecting joints to obtain a DNA library;

(A2) and selecting the fragment with the free DNA fragment size of 60-140bp from the DNA library to obtain a sequencing library, wherein the sequencing library is a processed sample.

3. A method for increasing the detection rate of pathogens in plasma free DNA, comprising the steps of:

(A1) extracting free DNA from blood plasma to be detected, performing end repair and adding A, and connecting joints to obtain a DNA library;

(A2) selecting the fragment with the size of the free DNA fragment of 60-140bp from the DNA library to obtain a sequencing library;

(A3) and performing on-machine sequencing on the sequencing library, and obtaining a detection result of the pathogen in the blood plasma to be detected from a sequencing result.

4. A system for detecting pathogens of bloodstream infections by plasma free DNA comprising

(B1) Reagents and/or apparatus for extracting free DNA from plasma;

(B2) reagents and/or instruments for end-repairing and adding A to free DNA and ligating linkers to obtain a DNA library;

(B3) (ii) reagents and/or apparatus capable of selecting fragments from the DNA library having an free DNA fragment size of 60-140bp as a sequencing library;

(B4) and the device can be used for performing on-machine sequencing on the sequencing library and obtaining the detection result of the pathogen in the plasma to be detected from the sequencing result.

5. The system of claim 4, wherein: in the step (B4), a conclusion output module is provided in the apparatus;

the conclusion output module is used for outputting the conclusion as follows: if the sequencing result contains specific sequence information of a certain pathogen, the to-be-detected plasma contains or is candidate to contain the corresponding pathogen; if the sequencing result does not contain specific sequence information of a certain pathogen, the plasma to be tested does not contain or does not contain a candidate corresponding pathogen.

6. A method for increasing the detection rate of pathogens in plasma free DNA, comprising the steps of:

(C1) extracting free DNA from blood plasma to be detected;

(C2) performing PCR amplification by using the free DNA as a template and adopting a pathogen specific primer pair; the size of the target fragment amplified by the primer pair is 60-140 bp;

(C3) and obtaining the detection result of the pathogen in the blood plasma to be detected according to the PCR amplification result.

7. Use of the system of claim 4 or 5 for the preparation of a product for the detection of pathogens of bloodstream infections by plasma free DNA.

8. Use of the system according to claim 4 or 5 and a readable medium carrying the method according to claim 3 for the preparation of a product for the detection of pathogens of bloodstream infections by plasma free DNA; or

Use of reagents and/or instruments for PCR amplification of plasma free DNA together with readable media describing the method according to claim 6 for the preparation of a product for detection of pathogens of bloodstream infections by plasma free DNA.

9. The method or system or use according to any one of claims 1-8, wherein: the size is 60-140bp, and the size is 64-132 bp; and/or

The pathogen is selected from the following: escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, enterococcus faecium, and/or Acinetobacter baumannii.

10. Use of the profile of pathogen free DNA in plasma for any of:

(D1) the development of products for the detection of blood stream-infecting pathogens by plasma free DNA;

(D2) the development of products for the diagnosis of infectious diseases by plasma free DNA;

(D3) the performance of the existing product for detecting the blood stream infection pathogen through the plasma free DNA is improved;

(D4) pathogen mock samples in plasma free DNA were prepared.

Technical Field

The invention relates to the technical field of biology, in particular to a plasma free DNA marker for diagnosing blood stream infection pathogens.

Background

Plasma-free DNA is DNA that is free from cells. It is currently believed that the free DNA source in the blood is associated with apoptosis, necrosis and active secretion of DNA by cells. Under normal physiological conditions, apoptotic and necrotic cells are rapidly eliminated, and therefore the concentration of free DNA in healthy human blood is low. In case of malignant tumor, organ transplantation, infection, etc., the released free nucleic acid is relatively high and cannot be effectively eliminated by the body, so that the concentration of the free nucleic acid in the blood plasma is increased.

Bloodstream infections are infections caused by the invasion of the bloodstream by a variety of pathogenic microorganisms, including bacteria, viruses, fungi, and the like. When blood stream infection occurs, pathogens are subjected to reproductive metabolism in blood or phagocytosed by leukocytes, and intracellular DNA is released into blood as plasma free DNA after cells are destroyed, so that both free DNA of human origin and free DNA of pathogens exist in plasma. For human free DNA, relatively comprehensive research is currently carried out, and it is believed that in the process of apoptosis or necrosis of human cells, genomic DNA will be intertwined with nucleosomes, and under the influence of the size of the nucleosomes, the human cells present a DNA fragment distribution of about 166bp in plasma; for pathogen free DNA, their release and degradation mechanisms in blood are currently less studied.

Currently, there are several companies at home and abroad that perform detection of pathogens of bloodstream infection by plasma free DNA. Taking Karius test of Karius corporation in the United states as an example, the method detects all free DNA in plasma in a metagenome second-generation sequencing mode, and the detected data is compared with a human source database. Removing the human source nucleic acid sequence, comparing the residual DNA sequence with the pathogen database, and judging the type of the infection pathogen according to the comparison result. However, this method detects a large amount of human-derived nucleic acid sequences during the detection process, and this part of the sequences needs to be removed during the information analysis process and belongs to "invalid" sequences, so that a large amount of data is wasted in sequencing data, and the detection sensitivity of pathogenic nucleic acids is also affected by a large amount of human-derived sequences. In practical use, the detection performance is low, the detection cost is high, and large-scale popularization is limited.

Detection of plasma free DNA pathogens based on PCR technology. The method carries out pathogen detection on the free DNA extracted from the plasma sample by designing a pathogen specific primer sequence and a probe, and has the advantages of simple operation, short detection period and the like. However, in the detection process, the method often has high design blindness due to no corresponding design guide scheme for the size of the amplified fragment product, and a large number of primers need to be screened and determined in practical application.

Disclosure of Invention

The invention establishes the size characteristics of free DNA fragments in blood plasma aiming at several common pathogen types of blood stream infection, can be used as a specific biomarker for diagnosing blood stream infection, and can guide the existing detection products to be further optimized by the characteristics so as to improve the detection performance.

In a first aspect, the invention claims a method for processing plasma free DNA.

The method for processing the free DNA of the blood plasma, which is claimed by the invention, can comprise the following steps: fragments of 60-140bp in size were selected from the plasma free DNA to be tested.

Further, the method can be used as a sample pretreatment method for detecting blood stream infection pathogens by plasma free DNA.

Further, the method can be used as a method for removing human-derived free DNA from plasma free DNA.

Furthermore, the method can be used as a sample pretreatment method for detecting blood stream infection pathogens by using a high-throughput sequencing technology through plasma free DNA.

In a second aspect, the invention claims a method for sample pretreatment for detection of blood stream infectious pathogens by plasma free DNA.

The sample pretreatment method for detecting blood stream infection pathogens through plasma free DNA, which is claimed by the invention, can comprise the following steps:

(A1) extracting free DNA from blood plasma to be detected, performing end repair and adding A, and connecting joints to obtain a DNA library;

(A2) and selecting the fragment with the free DNA fragment size of 60-140bp from the DNA library to obtain a sequencing library, wherein the sequencing library is a processed sample.

The method is a non-disease diagnostic treatment method.

In a specific embodiment of the invention, the size of the fragment selected from the DNA library in step (A2) is about 150-230bp (the adaptor sequence is about 90bp, and the size of the target free DNA fragment is about 60-140 bp).

Further, the sequencing library (processed sample) prepared by the method is suitable for detecting blood stream infection pathogens by using a high-throughput sequencing technology.

In a third aspect, the invention claims a method for increasing the detection rate of pathogens in plasma free DNA.

The method for improving the detection rate of pathogens in free DNA of blood plasma (high-throughput sequencing method) claimed by the invention can comprise the following steps:

(A1) and extracting free DNA from the plasma to be detected, performing end repair and A addition, and connecting joints to obtain a DNA library.

In this step, after the end repair, the 3 'end of the nucleic acid fragment is filled in by an enzyme reaction, and simultaneously, the 3' end is added with an A base to form a cohesive end.

In this step, after the linker is connected, a step of magnetic bead purification (such as XP magnetic bead) may be further included to remove the residual small fragment linker sequence.

(A2) And selecting the fragments with the free DNA fragment size of 60-140bp from the DNA library to obtain a sequencing library.

In this step, a step of library expansion is further included before the fragment selection is performed. In particular, PCR amplification can be performed in order to amplify the library signal and obtain a large number of library sequences.

In this step, fragment selection can be performed by magnetic bead purification. In the embodiment of the invention, the method is realized by the following steps: adding 0.7 times volume of XP magnetic beads (adsorbing fragments of more than about 230 bp) into the amplified DNA library, placing the DNA library on a magnetic frame, and then adding 1.2 times volume of XP magnetic beads (adsorbing fragments of more than 150 bp) into the supernatant (mainly fragments of less than 230 bp), wherein the fragments adsorbed by the magnetic beads are mainly nucleic acids of between 150 and 230bp (the length of the nucleic acids is the length after a sequencing joint is added, and the size of corresponding inserted fragments is between about 60 and 140bp after the joint sequence is removed).

(A3) And performing on-machine sequencing on the sequencing library, and obtaining a detection result of the pathogen in the blood plasma to be detected from a sequencing result.

In the step, the human sequence in the sequencing data is removed, and the remaining sequence is compared with a pathogen sequence library to obtain a pathogen comparison result.

The method is a non-disease diagnostic treatment method. Such as for testing blood products for eligibility, for containing a pathogen, or for infection by a pathogen.

In a specific embodiment of the invention, the size of the fragment selected from the DNA library in step (A2) is about 150-230bp (the adaptor sequence is about 90bp, and the size of the target free DNA fragment is about 60-140 bp).

In a fourth aspect, the invention claims a system for detecting blood stream infectious pathogens by plasma free DNA.

The system for detecting blood stream infection pathogens by plasma free DNA (applicable to high-throughput sequencing method) claimed by the invention can comprise

(B1) Reagents and/or apparatus for extracting free DNA from plasma;

(B2) reagents and/or instruments for end-repairing and adding A to free DNA and ligating linkers to obtain a DNA library;

(B3) (ii) reagents and/or apparatus capable of selecting fragments from the DNA library having an free DNA fragment size of 60-140bp as a sequencing library;

in a specific embodiment of the invention, the size of the fragment selected from the DNA library is about 150-230bp (the adaptor sequence is about 90bp, and the size of the target free DNA fragment is about 60-140 bp).

In a specific embodiment of the present invention, the reagent in (B3) is specifically XP magnetic beads.

(B4) And the device can be used for performing on-machine sequencing on the sequencing library and obtaining the detection result of the pathogen in the plasma to be detected from the sequencing result.

Further, in the step (B4), a conclusion output module is provided in the apparatus.

The conclusion output module is used for outputting the conclusion as follows: if the sequencing result (i.e. the sequencing result of the sequencing library consisting of the target fragments, which are target free DNA fragments connected with the adapters, in the embodiment of the present invention, the adapter sequence is about 90bp, and the size of the target free DNA fragments is about 60-140bp) contains specific sequence information of a certain pathogen, the plasma to be tested contains or is candidate to contain the corresponding pathogen; if the sequencing result does not contain specific sequence information of a certain pathogen, the plasma to be tested does not contain or does not contain a candidate corresponding pathogen.

In a fifth aspect, the invention claims a method for increasing the detection rate of pathogens in plasma free DNA.

The method for improving the pathogen detection rate in the free DNA of the blood plasma, which is claimed by the invention, can comprise the following steps:

(C1) extracting free DNA from blood plasma to be detected;

(C2) performing PCR amplification by using the free DNA as a template and adopting a pathogen specific primer pair; the size of the target fragment amplified by the primer pair is 60-140 bp;

(C3) and obtaining the detection result of the pathogen in the blood plasma to be detected according to the PCR amplification result.

The method is a non-disease diagnostic treatment method. Such as for testing blood products for eligibility, for containing a pathogen, or for infection by a pathogen.

In a sixth aspect, the invention claims the use of a system according to the fourth aspect above for the preparation of a product for the detection of pathogens of bloodstream infections by plasma free DNA.

In a seventh aspect, the invention claims the use of a system according to the fourth aspect above and a readable medium carrying the method according to the third aspect above for the preparation of a product for the detection of pathogens of bloodstream infections by plasma free DNA.

In an eighth aspect, the invention claims reagents and/or instruments for PCR amplification of plasma free DNA and the use of a readable medium carrying the method according to the sixth aspect as described above for the preparation of a product for detection of pathogens of bloodstream infections by plasma free DNA.

In the above aspects, the size of 60 to 140bp may specifically be 64 to 132 bp.

Accordingly, the pathogen may be selected from the following: escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, enterococcus faecium, and/or Acinetobacter baumannii.

In a ninth aspect, the invention claims the use of the profile of pathogen free DNA in plasma for any of:

(D1) the development of products for the detection of blood stream-infecting pathogens by plasma free DNA;

(D2) the development of products for the diagnosis of infectious diseases by plasma free DNA;

(D3) the performance of the existing product for detecting the blood stream infection pathogen through the plasma free DNA is improved;

(D4) pathogen mock samples in plasma free DNA were prepared.

In the present invention, the pathogen may be selected from the following: escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, enterococcus faecium, and/or Acinetobacter baumannii. Accordingly, the peak distribution of the pathogen free DNA in plasma ranged from 64 to 132 bp. More specifically, the peak value of the free DNA distribution of the Escherichia coli in the plasma is 89bp, the peak value of the free DNA distribution of the Klebsiella pneumoniae in the plasma is 132bp, the peak value of the free DNA distribution of the Staphylococcus aureus in the plasma is 64bp, the peak value of the free DNA distribution of the Acinetobacter baumannii in the plasma is 112bp, and the peak value of the free DNA distribution of the enterococcus faecium in the plasma is 118 bp.

The invention is based on metagenome sequencing technology, researches the characteristics of free DNA in plasma of patients infected by different pathogens, and establishes a specific pathogen molecular marker for diagnosis of blood stream infection.

(1) The invention researches the distribution range of free nucleic acid in plasma samples infected by Escherichia coli, Klebsiella pneumoniae, staphylococcus aureus, enterococcus faecium and Acinetobacter baumannii, and determines the distribution of the free nucleic acid fragments of the 5 bacteria in the plasma.

(2) Based on the distribution characteristics of the pathogen free nucleic acid fragments discovered by the invention, the pathogen free nucleic acid can be specifically screened by a fragment selection technology or the human source nucleic acid can be specifically removed, so that the sensitivity of pathogen detection in plasma macro genome detection is improved.

(3) According to the sizes of free DNA fragments of different pathogens in plasma, the length range of PCR amplification product fragments can be guided to be designed so as to improve the detection sensitivity of PCR.

(4) The invention can also provide basis for preparing pathogen simulation samples in the development process of plasma free DNA detection products.

The beneficial effects of the invention are as follows:

1. the invention discovers a pathogen specific molecular marker in blood plasma, establishes the characteristics of the marker, can be used for diagnosing patients with blood stream infection, has obviously better diagnosis timeliness than the traditional blood culture method, and can obtain a detection result in a short time.

The pathogen specific molecular marker established by the invention is pathogen free DNA existing in blood plasma, and when blood stream infection occurs, free DNA released by human apoptosis and free DNA released by infection pathogens exist in the blood plasma of a patient. According to the characteristics of the pathogen free DNA established in the invention (see example 1), aiming at the fragment size distribution of different pathogen free DNAs in plasma, the detection of the corresponding pathogen can be realized by detecting the free DNA in a specific distribution range, the detection of all the free DNA in the specific size in the plasma can be realized by a metagenome secondary sequencing method, and the diagnosis of the infectious pathogen can be realized by identifying the pathogen nucleic acid sequence contained in the free DNA by a bioinformatics comparison method. The method can be used for simultaneously detecting tens of thousands of infectious pathogens. In addition, for a patient suspected of being infected by a specific pathogen type, in order to obtain the etiology evidence as soon as possible, primers with corresponding amplified fragment sizes can be designed by referring to the fragment distribution peak value of different free DNAs in plasma established in the invention so as to improve the amplification success rate, and the diagnosis is carried out by a PCR method. The free DNA in the blood plasma is extracted, specific pathogen primers are adopted for PCR amplification or fluorescence PCR detection, whether infection of corresponding pathogens exists or not is rapidly judged according to the detection result, and a basis is provided for rapid diagnosis of blood stream infection. The invention utilizes free DNA in plasma to carry out molecular diagnosis, avoids the influence of interference substances such as hemoglobin and the like on molecular detection when a whole blood sample is used, and simultaneously can realize high-efficiency detection of infectious pathogens according to the characteristics of the molecular marker established by the invention.

2. The invention researches the distribution of the pathogenic molecule marker fragments in the blood plasma, and can guide the design of related detection products and improve the detection performance according to the specificity of the fragment distribution.

The present inventors studied the distribution pattern of 5 pathogens in plasma free DNA, which are common to bloodstream infections (see example 1). Wherein the free DNA distribution peak value of the Escherichia coli in plasma is 89bp, and the distribution median is 116 bp; the free DNA distribution peak value of the Klebsiella pneumoniae in plasma is 132bp, and the distribution median is 166 bp; the free DNA distribution peak value of staphylococcus aureus in plasma is 64bp, the distribution median is 103bp, the free DNA distribution peak value of acinetobacter baumannii in plasma is 112bp, the distribution median is 128bp, the free DNA distribution peak value of enterococcus faecium in plasma is 118bp, and the distribution median is 134 bp. Compared with the different pathogens, the human source free DNA in the plasma has a distribution peak value of 166bp and a distribution median of 165bp, the difference between the peaks of the different pathogens and the human source free DNA is 34-102bp, and the fragment distribution peak shapes of the 5 pathogens in the plasma can be judged to show a trailing distribution according to the peak values and the median sizes of the different pathogen free DNA. According to the distribution difference, free DNA distributed in a specific interval can be screened for detection so as to improve the detection sensitivity of corresponding pathogens. For example, enrichment detection is performed on 60-140bp fragments in plasma by a fragment selection method, and pathogen comparison is performed on the result after metagenome second-generation sequencing, so that the detection of the Escherichia coli nucleic acid sequence can be effectively improved (see example 2). Therefore, the pathogen free DNA related characteristics established by the invention can guide the design of high-performance detection products and improve the detection of infectious pathogen nucleic acid.

3. The invention establishes the distribution form of different pathogens in the free DNA of the plasma after infection, and provides a basis for the preparation of the plasma simulation sample in the product development.

The invention establishes the free DNA distribution of 5 common pathogens of blood stream infection in blood plasma, including Escherichia coli, Klebsiella pneumoniae, staphylococcus aureus, Acinetobacter baumannii and enterococcus faecium. The free DNA distribution of different pathogens in plasma is mainly concentrated between 64-132bp, while the distribution of human free DNA in plasma is 166 bp.

At present, with the development of molecular biology technology, plasma free DNA has been applied in many diagnostic and screening fields. Products for pathogen diagnosis through plasma free DNA have also appeared in succession, but the declared detection performance of products from different manufacturers has certain difference, the great reason is that there is no relevant research on the characteristics of pathogen free DNA at present, and different manufacturers do not consider the real distribution of pathogen free DNA in plasma during the evaluation of product performance, so that the declared performance may not be in accordance with practical application. The free DNA distribution of different pathogens established by the invention can guide the design and performance evaluation of related pathogen detection products, the free DNA of the corresponding pathogen is prepared according to the size of a specific fragment, the free DNA is added into a plasma sample to prepare a simulation sample for detection, the detection performance of the product is evaluated, and support is provided for the accuracy of the product performance.

Drawings

FIG. 1 shows the distribution of human nucleic acids in plasma samples of different infection types.

FIG. 2 shows the peaks of the distribution of nucleic acids of different pathogens in plasma.

FIG. 3 is the median distribution of nucleic acids from different pathogens in plasma.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific reagents used in the following examples and the nucleic acid sequences used are shown in tables 1 and 2.

TABLE 1 reagent name and manufacturer

Name of reagent	Manufacturer of the product
		Nucleic acid purification reagent	BGI SHENZHEN CO., Ltd.
T4 DNA Polymerase	Enzymatics
		T4 PNK	Enzymatics
10×T4 PNK buffer	Enzymatics
		rTaq	TaKaRa
dATP(100mM)	Enzymatics
		dNTP(25mM)	Enzymatics
ATP(100mM)	Thermo Fisher
		T4 DNA Ligase	Enzymatics
50％PEG 8000	Rigaku
		KaPa HiFi Ready Mix	Kapa biosystems

TABLE 2 linker sequences and primer sequences