CN118127187A - Respiratory tract pathogenic microorganism detection kit based on targeted sequencing and application thereof - Google Patents

Abstract

The application belongs to the technical field of biological detection, and particularly discloses a respiratory tract pathogenic microorganism detection kit and method based on targeted sequencing and application thereof, wherein the kit can simultaneously amplify 36 respiratory tract pathogenic microorganism target genes in one reaction tube, and can detect clinical common pathogenic microorganisms possibly existing in a sample by combining a high-throughput sequencing technology, and has the advantages of high coverage, high sensitivity and the like.

Description

Respiratory tract pathogenic microorganism detection kit based on targeted sequencing and application thereof

Technical Field

The application belongs to the technical field of biological detection, and particularly relates to a respiratory tract pathogenic microorganism detection kit based on targeted sequencing and a detection method thereof.

Technical Field

The common pathogenic microorganisms in respiratory tract mainly comprise viruses, bacteria, mycoplasma, chlamydia, rickettsiae and the like, and more than 90% of acute respiratory tract infections are statistically caused by the viruses. A pathogenic microorganism may cause a variety of clinical symptoms, and the same clinical manifestation may be caused by a variety of pathogenic microorganisms. The probability of bacterial infection of respiratory tract is lower than that of virus, but once the infection is caused, symptoms are generally serious and positioning is obvious, such as tonsillitis, tracheitis, nasosinusitis and the like. Viruses include EBV, CMV, HSV, HHV, bocavirus, adenovirus, rhinovirus, coronavirus, respiratory syncytial virus, influenza virus, parainfluenza virus, and the like.

Compared with the traditional pathogen detection technology based on separation culture, the second generation sequencing (mNGS) of the metagenomics does not need to separate and culture pathogens, does not depend on known nucleic acid sequences, can detect various microorganisms (such as viruses, bacteria, fungi, parasites and the like) in an unbiased and full-coverage way, does not need specific amplification, greatly saves detection time, improves diagnosis efficiency, and has incomparable advantages in identifying unknown species and pathogens which are difficult to culture. However, mNGS is a non-targeted detection, so long as the nucleic acid in the sample can be detected, so if a large amount of extracted nucleic acid exists in the sample, the trace pathogenic nucleic acid can be submerged under a huge host background, and false negative results cannot be detected, and therefore, efficient host elimination of the sample with high host content is an important link to be considered in mNGS.

Pathogen targeted sequencing (targetted Next-Generation Sequencing, tNGS) can detect tens to hundreds of known pathogenic microorganisms and virulence and/or drug resistance genes thereof in a sample to be detected by combining two technologies of super multiplex PCR amplification and high throughput sequencing. Compared with pathogen metagenome sequencing (mNGS), tNGS has the advantages of definite pathogen spectrum range, low sequencing cost and the like.

Disclosure of Invention

In order to solve the technical problems, the application provides a respiratory tract pathogenic microorganism detection system based on targeted sequencing, a kit and application thereof.

Specifically, the technical scheme adopted by the application is as follows:

The application firstly provides a primer group for detecting respiratory tract pathogenic microorganisms, wherein the primer is aimed at bacterial pathogens, fungal pathogens and viral pathogens.

Further, the bacterial pathogen includes: streptococcus pneumoniae, legionella pneumophila, staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, mycobacterium tuberculosis complex, pertussis baupecia, corynebacterium diphtheriae, burkholderia melitensis and francissia tularensis; the fungus pathogens include: coccidioidomycosis, paracoccidiomycosis brasiliensis, candida albicans, blastomyces dermatitis, histoplasmosis capsulata, cryptococcus neoformans, cryptococcus garteus, cercospora spinosa; the viral pathogens include: influenza virus, parainfluenza virus, adenovirus, human respiratory syncytial virus a, human respiratory syncytial virus B, rhinovirus, human metapneumovirus, coronavirus NL63, coronavirus HKU1, coronavirus 229E.

Further, the primer is also directed to a drug resistance gene; the drug resistance gene comprises: mecA, NDM-1, KPC-2, OXA-48, VIM and mcr-1.

Further, the primer sequence comprises a sequence shown as SEQ ID NO.1-72 or has more than 80% homology with the sequence shown as SEQ ID NO. 1-72.

Further, in the primers, the 5' end of each forward amplification primer is connected to 5'-CTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3'; connecting; the 5' end of each reverse amplification primer is ligated 5'-TTCCTAAGACCGCTTGGCCTCCGACTT-3'.

The application also provides a kit or a product, which is characterized by comprising the primer group.

Further, the kit or product further comprises a PCR buffer and a DNA polymerase; preferably, the kit can also comprise a sample DNA/RNA co-extraction reagent and a reverse transcription reagent.

The application also provides application of the primer group in preparation of respiratory tract pathogenic microorganism detection reagents.

Further, the application comprises the following steps:

s1, obtaining nucleic acid of a sample to be detected;

s2, performing reverse transcription by taking the nucleic acid sample obtained in the step S1 as a template to obtain cDNA;

S3, carrying out multiplex PCR (polymerase chain reaction) amplification by using the primer respectively by taking DNA in the sample and cDNA obtained by reverse transcription as templates, and preparing a product mixture from a DNA amplification product and a cDNA amplification product according to a certain proportion;

s4, using the product mixed solution obtained in the step S3 as a template, performing additive joint PCR amplification by using a joint sequence primer pair, and adding a sequencing joint sequence to a multiplex PCR amplified product to obtain a sequencing library;

S5, performing high-throughput sequencing on the sequencing library obtained in the step S4 to obtain a sequencing sequence;

S6, comparing the sequencing sequence with a pathogenic microorganism target sequence database, and counting and accurately comparing the number of reads of a specific pathogenic microorganism target;

s7, calculating the standardized reads number of the pathogenic microorganisms by using the following formula:

wherein, RPM:1M reads pathogenic microorganism detection number; pathogen reads number number of reads detected by pathogen; CLEAN READS number of valid reads after sample pretreatment

The application also provides a method for detecting respiratory tract pathogenic microorganisms, which comprises the following steps:

s1, obtaining nucleic acid of a sample to be detected;

s2, performing reverse transcription by taking the nucleic acid sample obtained in the step S1 as a template to obtain cDNA;

S3, performing multiplex PCR (polymerase chain reaction) amplification by using the primer respectively by taking DNA in a sample and cDNA obtained by reverse transcription as templates, and preparing a product mixture from a DNA amplification product and a cDNA amplification product according to a certain proportion;

s4, using the product mixed solution obtained in the step S3 as a template, performing additive joint PCR amplification by using a joint sequence primer pair, and adding a sequencing joint sequence to a multiplex PCR amplified product to obtain a sequencing library;

S5, performing high-throughput sequencing on the sequencing library obtained in the step S4 to obtain a sequencing sequence;

S6, comparing the sequencing sequence with a pathogenic microorganism target sequence database, and counting and accurately comparing the number of reads of a specific pathogenic microorganism target;

s7, calculating the standardized reads number of the pathogenic microorganisms by using the following formula:

Wherein, RPM:1M reads pathogenic microorganism detection number; pathogen reads number number of reads detected by pathogen; CLEAN READS number of valid reads after sample preprocessing.

The application has the beneficial technical effects that:

1) The application obtains a high-weight amplification primer group system and a kit and the like suitable for targeted sequencing of respiratory tract pathogenic microorganisms through repeated optimization design, wherein the high-weight system can amplify 36 pathogenic microorganism target genes simultaneously in one reaction tube, and can detect clinical common pathogenic microorganisms possibly existing in a sample efficiently, sensitively, specifically and accurately by combining a high-throughput sequencing technology.

2) The method has the advantage of high coverage: the application can cover 36 common pathogenic microorganisms in clinic, including bacteria, fungi, viruses, mycoplasma, chlamydia, and the like; aiming at clinical suspected infection samples, the accurate diagnosis and treatment scheme can be formulated by detecting common pathogenic microorganisms in the clinical suspected infection samples and assisting in clinically and accurately identifying common pathogens.

3) The application can detect drug-resistant genes besides identifying pathogenic microorganisms, and the primer system of the application covers the drug-resistant gene sequence, and can detect whether the drug-resistant genes exist in a sample by sequencing analysis.

4) The application can detect DNA and RNA viruses at the same time, and the sample treatment of the application covers common bacteria, fungi, parasites, mycoplasma, chlamydia and DNA viruses which take DNA as genetic materials, and RNA viruses which take RNA as genetic materials, such as respiratory syncytial virus, influenza virus, parainfluenza virus, epstein-Barr virus, coxsackie virus, rhinovirus and the like.

Drawings

FIG. 1 shows amplification effects before and after optimization of Candida albicans primers. Wherein, the left graph is the analysis result of the amplification products of the two pairs of primers before the primer is not optimized; the right panel shows the results of two pairs of primer amplification product analysis after primer optimization.

FIG. 2, QPCR single-screen amplification curve and dissolution curve before primer optimization;

FIG. 3, QPCR single-screen amplification curve and dissolution curve after primer optimization;

FIG. 4 shows the results of electrophoresis detection of the products before and after optimization of the multiplex amplification system.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Partial term definition

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the application are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

The term "about" in the present application means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operation in other sequences than described or illustrated herein.

The primer group for detecting respiratory tract pathogenic microorganisms is aimed at bacterial pathogens, fungal pathogens and viral pathogens.

In some embodiments, the bacterial pathogen comprises: streptococcus pneumoniae, legionella pneumophila, staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, mycobacterium tuberculosis complex, pertussis baupecia, corynebacterium diphtheriae, burkholderia melitensis and francissia tularensis; the fungus pathogens include: coccidioidomycosis, paracoccidiomycosis brasiliensis, candida albicans, blastomyces dermatitis, histoplasmosis capsulata, cryptococcus neoformans, cryptococcus garteus, cercospora spinosa; the viral pathogens include: influenza virus, parainfluenza virus, adenovirus, human respiratory syncytial virus a, human respiratory syncytial virus B, rhinovirus, human metapneumovirus, coronavirus NL63, coronavirus HKU1, coronavirus 229E.

In some embodiments, the primer is also directed to a drug resistance gene; the drug resistance gene comprises: mecA, NDM-1, KPC-2, OXA-48, VIM, mcr-1.

In some embodiments, the primer sequences include the sequences set forth in SEQ ID NOS.1-72, or have greater than 80% homology to the sequences set forth in SEQ ID NOS.1-72.

In some more specific embodiments, the 5' end of each forward amplification primer is ligated 5'-CTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3' of the primers; the 5' end of each reverse amplification primer is ligated 5'-TTCCTAAGACCGCTTGGCCTCCGACTT-3'.

The kit or the product of the application comprises the primer group. In some specific embodiments, the kit or product further comprises a kit comprising a PCR buffer and a DNA polymerase; in some more specific embodiments, sample DNA/RNA co-extraction reagents, reverse transcription reagents may also be included.

The primer group of the application is applied to the preparation of respiratory tract pathogenic microorganism detection reagent; or in the detection of pathogenic microorganisms of the respiratory tract

In some specific embodiments, the application comprises the steps of:

s1, obtaining nucleic acid of a sample to be detected;

s2, performing reverse transcription by taking the nucleic acid sample obtained in the step S1 as a template to obtain cDNA;

S3, carrying out multiplex PCR (polymerase chain reaction) amplification by using the primer respectively by taking DNA in the sample and cDNA obtained by reverse transcription as templates, and preparing a product mixture from a DNA amplification product and a cDNA amplification product according to a certain proportion;

s4, using the product mixed solution obtained in the step S3 as a template, performing additive joint PCR amplification by using a joint sequence primer pair, and adding a sequencing joint sequence to a multiplex PCR amplified product to obtain a sequencing library;

S5, performing high-throughput sequencing on the sequencing library obtained in the step S4 to obtain a sequencing sequence;

S6, comparing the sequencing sequence with a pathogenic microorganism target sequence database, and counting and accurately comparing the number of reads of a specific pathogenic microorganism target;

s7, calculating the standardized reads number of the pathogenic microorganisms by using the following formula:

wherein, RPM:1M reads pathogenic microorganism detection number; pathogen reads number number of reads detected by pathogen; CLEAN READS number of valid reads after sample pretreatment

The method for detecting the respiratory tract pathogenic microorganisms comprises the following steps:

s1, obtaining nucleic acid of a sample to be detected;

s2, performing reverse transcription by taking the nucleic acid sample obtained in the step S1 as a template to obtain cDNA;

S3, performing multiplex PCR (polymerase chain reaction) amplification by using the primer respectively by taking DNA in a sample and cDNA obtained by reverse transcription as templates, and preparing a product mixture from a DNA amplification product and a cDNA amplification product according to a certain proportion;

s4, using the product mixed solution obtained in the step S3 as a template, performing additive joint PCR amplification by using a joint sequence primer pair, and adding a sequencing joint sequence to a multiplex PCR amplified product to obtain a sequencing library;

S5, performing high-throughput sequencing on the sequencing library obtained in the step S4 to obtain a sequencing sequence;

S6, comparing the sequencing sequence with a pathogenic microorganism target sequence database, and counting and accurately comparing the number of reads of a specific pathogenic microorganism target;

s7, calculating the standardized reads number of the pathogenic microorganisms by using the following formula:

Wherein, RPM:1M reads pathogenic microorganism detection number; pathogen reads number number of reads detected by pathogen; CLEAN READS number of valid reads after sample preprocessing.

The application is illustrated below in connection with specific embodiments.

Example 1 selection and establishment of pathogenic bacteria

In order to realize comprehensive and effective detection of respiratory pathogens, the application analyzes the types and the types of common respiratory pathogens, combines the feasibility of a multiplex amplification system, and finally determines the targeting species and drug resistance genes as follows:

wherein, the pathogen against bacteria is shown in the following table:

The pathogen against fungi is shown in the following table;

Sequence number	Pathogenic chinese name	Pathogenic english name
			1	Coccidioidomycosis (Bl.) Sp	Coccidioides immitis
2	Sporoborium besii	Coccidioides posadasii
			3	Paracoccus Brazil	Paracoccidioides brasiliensis
4	Candida albicans	Candida albicans
			5	Acidovorax dermatitis (Bv.) Kuntze	Blastomyces dermatitidis
6	Histoplasma capsulatum (Bt)	Histoplasma capsulatum
			7	Marneffei basket	Talaromyces marneffei
8	Cryptococcus neoformans	Cryptococcus neoformans
			9	Cryptococcus garteus	Cryptococcus gattii
10	Saidosporine tip	Scedosporium apiospermum

The viral pathogens aimed at are shown in the following table

The specific drug resistance genes are shown in the following table:

Sequence number	Drug resistance gene name	Encoded protein name
			1	mecA	Methicillin-resistant gene
2	NDM-1	New Deril metal-beta-lactamase coding gene
			3	KPC-2	Carbapenemase encoding gene
4	OXA-48	Carbapenemase encoding gene
			5	VIM	Metal-beta-lactamase coding gene
6	mcr-1	Colistin resistance gene

Example 2, targeting region screening and primer design

1) Screening process for targeting regions

After determining the types of pathogenic microorganisms, the specificity and the sensitivity in the multiplex detection process are realized, and when the targeting primer is designed, the logic of the targeting region can reach more than 95 percent, and the coverage reaches more than 95 percent; in addition, the application also gives consideration to drug resistance detection, and part of the primer targeting region also comprises a drug resistance locus region.

An exemplary display portion primer sequence optimization procedure is as follows.

Since the targeted regions are more, the application is shown by specific examples of the optimization of the targeted regions by using only 3 groups of primers:

The application realizes remarkable improvement in detection specificity and coverage after optimization and adjustment of coverage area.

2) Designing, screening and optimizing the primer sequence:

In view of the high heavy primer system, the primer design requirements of the application are strict, and meanwhile, a plurality of factors need to be considered, and the whole primer undergoes the following steps:

1) Preliminary design of the super multiplex PCR primers by using primer design software or a primer design website; the requirements are: the length of the primer amplicon is 100-600bp, the GC content of the sequence is 45-65%, the length of the primer is 17-25bp, no more than 5 bases are strictly complementary between the two primers, and the primers have no multimeric sequence of continuously more than 5 bases;

2) Performing primer screening based on raw letter analysis to obtain a double-screening primer with high specificity and low dimer;

3) Screening a super-multiplex PCR primer with high amplification efficiency through a wet experiment, and adjusting a specific primer sequence;

4) And (5) performing primer evaluation re-screening based on the electrophoresis gray scale evaluation system and the QPCR evaluation system.

In addition, when the primers of the application are used, the 5' end of each forward amplification primer is connected with a forward universal primer 5'-CTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3'; the 5' end of each reverse amplification primer is ligated to a reverse universal primer 5'-TTCCTAAGACCGCTTGGCCTCCGACTT-3'. Wherein the forward amplification primer is a sequence which recognizes a pathogenic target and is amplified, and the forward universal primer and the reverse universal primer are MGI sequencing platform adaptor sequences.

Exemplary primer sequence optimizations are as follows:

a. Optimization of Single amplification

For candida albicans as an example, primer information during initial design and after optimization of the application is as follows:

Through detection of single primer amplification efficiency, the white primer amplification efficiency before optimization is lower, the problems of strip dispersion and the like (left in figure 1) exist, and the QPCR verification of single pair primers shows that the non-specific amplification of human gDNA (figure 2) exists, so that the mixed requirement of multiple PCRs can not be met. After the sequence is redesigned and optimized by the bioinformatic analysis, the amplification efficiency of the primer is obviously improved (right in fig. 1), only target specific amplification is carried out, non-specific amplification of the unmanned gDNA (fig. 3) is carried out, and the primer after adjustment has obvious amplification efficiency advantage.

B. Optimization of multiple systems

After individual screening, the mixed primer sets were subjected to multiplex amplification. For streptococcus pneumoniae, the streptococcus pneumoniae cannot be amplified in a multiple system during preliminary design; after primer set optimization, the primer set can be effectively amplified in a multiplex system, and the primer set is specifically shown in FIG. 4.

C. Optimization of dimers

Although the correlation between the individual primer sets has been avoided to the greatest extent by analysis in the present multiplex system and optimized by primer set screening of individual species, there is also a small fraction of dimers that have not been removed, and therefore primers that form more than 1% of dimers need to be found by analysis of such sequencing reads, deleted from the primer set (Mycobacterium_tuberculosis_ 1_F), re-analyzed and the latest primer set designed. Thereby finally reducing the possibility of dimer formation of the multiplex amplification primer group and finally improving the effective utilization rate of sequencing data. Specific analysis results for mycobacterium tuberculosis are as follows:

in summary, the optimization test shows the final multiple targeting primer system determined by the application as follows.

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Example 3 preparation of kit and detection System set-up

1. The kit of the application is established based on the primer amplification system, and comprises the primer system, PCR buffer solution, DNA polymerase and the like. The PCR buffer may be any buffer capable of performing PCR amplification, including, but not limited to dNTPs, mgCl ₂, tris-HCl, KCl, and (NH ₄)₂SO₄, etc. the kit may also include sample DNA/RNA co-extraction reagents, reverse transcription reagents, and the sample DNA/RNA extraction reagents may be any reagents, combination of reagents, or commercial kit capable of extracting sample DNA/RNA, such as, for example, reagents, combination of reagents, or commercial kit that utilize enzymatic and chemical reagents to cleave proteins in a sample, cleave cells, and thereby release nucleic acids, by enzymatic and chemical cleavage methods, wherein the enzymes include, but are not limited to, proteinase K, and the chemical reagents include, but are not limited to, guanidine isothiocyanate.

2. Establishing a target sequencing-based pathogenic microorganism detection method, which specifically comprises the following steps:

s1, obtaining nucleic acid of a sample to be detected;

The procedure uses ZYMO Quick-DNA/RNA ^TM visual Kit (D7020) for the extraction of the nucleic acids (DNA and RNA) of the sample to be tested. The operation steps are as follows:

(1) Preparing 0.5% beta-mercaptoethanol (V/V) and 80% absolute ethanol;

(2) Adding DNA/RNA SHIELD protective solution into the sample according to the ratio of 1:1 under the condition of room temperature (20-30 ℃);

(3) Adding 400 mu L VIRAL DNA/RNA Buffer into 400 mu L sample and mixing uniformly;

(2) Transferring the mixture to an adsorption Column Zymo-Spin ^TM IIC-XLR Column, placing in a collection tube, centrifuging for 2min at 14000g, and transferring the adsorption Column to a new collection tube;

(3) Adding 500 mu L VIRAL WASH Buffer into the adsorption column, centrifuging for 30sec at 12000g, discarding the waste liquid, and repeating the steps;

(4) 500. Mu.L of 80% absolute ethanol was added to the column and 12000g centrifuged for 1min to ensure complete removal of the eluent. Changing the new collection tube, shaking 12000g, centrifuging for 1min, carefully transferring the column into a new 1.5mL centrifuge tube without nuclease;

(5) Suspending and dripping 35 μl of DNase/RNase-FREE WATER (Ambion ^TM, invitrogen) into the middle of the adsorption membrane, standing at room temperature for 1min, centrifuging for 1min at 14000g, retaining eluent in 1.5mL centrifuge tube, and discarding the adsorption column;

(6) Taking 1 mu L of elution product to perform Qubit quantification (Thermo Fisher) and measuring the concentration of extracted nucleic acid; if the extraction concentration is 0, resampling and extraction are required.

S2, performing reverse transcription by taking the nucleic acid sample obtained in the step S1 as a template to obtain cDNA;

the procedure used the SuperScript ^TMVILO^TM CDNA SYNTHESIS KIT kit for cDNA synthesis of the nucleic acid to be tested. The specific operation steps are as follows:

(1) For a single reaction, the following components were mixed in a tube on ice. For multiple reactions, a master mix without RNA was prepared.

Component (A)	Volume (mu L)
		5X VILOTMReaction Mix	4
10X SuperScriptTMEnzyme Mix	2
		RNA(up to 2.5μg)	X
DEPC-treated water	Up to 20

(2) The tube contents were gently mixed and incubated at 25℃for 10 minutes.

(3) The tube was incubated at 42℃for 60 minutes.

(4) The reaction was terminated at 85℃for 5 minutes.

(5) The reaction product was either stored at-20℃or directly subjected to the next reaction.

S3, performing multiplex PCR (polymerase chain reaction) amplification by using the primer set in claim 2 respectively by taking DNA and cDNA as templates, and preparing a product mixture of a DNA amplification product and a cDNA amplification product according to a certain proportion;

the components were added to eight-tube for PCR amplification according to the following system:

Component (A)	Volume (mu L)
		Enzyme premix (Tiangen)	12.5
Primer set F	1.0
		Primer set R	1.0
Nucleic acid templates	10.0
		Nuclease-Free Water	0.5
Total	25.0

(2) The following procedure was set up on the PCR instrument for PCR amplification:

S4, using the product mixed solution obtained in the step S3 as a template, performing additive joint PCR amplification by using the joint sequence primer pair of claim 3, and adding a sequencing joint sequence to a multiplex PCR amplified product to obtain a sequencing library;

(1) The components were added to eight-tube for PCR amplification according to the following system:

Component (A)	Volume (mu L)
		Enzyme premix (Tiangen)	12.5
Universal primer Index for adapter (MGI)	2.0
		Step S3 product	10.0
Nuclease-Free Water	0.5
		Total	25.0

(2) The following procedure was set up on the PCR instrument for PCR amplification:

(3) The PCR product was subjected to magnetic bead (Agencout AMPure XP, beckman) purification and the final elution volume was 30. Mu.L.

S5, performing high-throughput sequencing on the sequencing library obtained in the step S4 to obtain a sequencing sequence;

The sequencing system used in the step is a MGISEQ-200RS sequencer manufactured by Hua Dazhi, and the sequencing is carried out on the machine according to the corresponding operation steps, and the sequencing sequence result is obtained after about 8 hours.

S6, comparing the sequencing sequence with a pathogenic microorganism target sequence database, and counting and accurately comparing the number of reads of a specific pathogenic microorganism target;

s7, calculating the standardized reads number of the pathogenic microorganisms by using the following formula:

Wherein, RPM:1M reads pathogenic microorganism detection number; pathogen reads number: number of reads detected by pathogen; CLEAN READS number: number of valid reads after sample pretreatment.

Example 4 evaluation of Limit Performance

In this example, performance was evaluated by selecting a representative of the typical pathogen for each species tested, bacterial selection was made for Pseudomonas aeruginosa (Pseudomonas aeruginosa), drug resistance gene selection was made for KPC-2 (from Klebsiella pneumoniae), fungi selection was made for Candida albicans (Candida albicans), and virus selection was made for human respiratory syncytial virus A (HSV_A) with the final results shown in the following table, and the minimum detection Limit (LOD) for all typical species could reach 10CFU/mL.

Example 5 clinical sample detection verification

In this example, 20 clinical samples of known pathogenic bacteria or drug resistance were collected, and detected and compared by the method of the present application, and the results are shown in the following table.

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It can be seen that, for 20 samples of different pathogens or drug resistance, the prediction accuracy of the method of the present application is very high, 100%, and at the same time, the method of the present application can also detect pathogens that cannot be detected by conventional standard methods, such as S14, S19, S20, etc.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solution of the present application, and not limiting thereof; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims (10)

1. A primer set for detecting respiratory tract pathogenic microorganisms, wherein the primer is directed against bacterial pathogens, fungal pathogens and viral pathogens.

2. The primer set of claim 1, wherein the bacterial pathogen comprises: streptococcus pneumoniae, legionella pneumophila, staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, mycobacterium tuberculosis complex, pertussis baupecia, corynebacterium diphtheriae, burkholderia melitensis and francissia tularensis; the fungus pathogens include: coccidioidomycosis, paracoccidiomycosis brasiliensis, candida albicans, blastomyces dermatitis, histoplasmosis capsulata, cryptococcus neoformans, cryptococcus garteus, cercospora spinosa; the viral pathogens include: influenza virus, parainfluenza virus, adenovirus, human respiratory syncytial virus a, human respiratory syncytial virus B, rhinovirus, human metapneumovirus, coronavirus NL63, coronavirus HKU1, coronavirus 229E.

3. The primer set of any one of claims 1-2, wherein the primer is further directed against a drug resistance gene; the drug resistance gene comprises: mecA, NDM-1, KPC-2, OXA-48, VIM and mcr-1.

4. A primer set according to any one of claims 1 to 3, wherein the primer sequence comprises the sequence shown in SEQ ID No.1 to 72 or has more than 80% homology with the sequence shown in SEQ ID No.1 to 72.

5. The primer set of claim 4, wherein the 5' end of each forward amplification primer is ligated 5'-CTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT-3' to each of the primers; the 5' end of each reverse amplification primer is ligated 5'-TTCCTAAGACCGCTTGGCCTCCGACTT-3'.

6. Kit or product, characterized in that it comprises a primer set according to any one of claims 1 to 5.

7. The kit or product of claim 5, further comprising a PCR buffer and a DNA polymerase; preferably, the kit can also comprise a sample DNA/RNA co-extraction reagent and a reverse transcription reagent.

8. Use of the primer set according to any one of claims 1 to 5 for preparing a respiratory tract pathogenic microorganism detection reagent.

9. The application according to claim 8, characterized in that it comprises the steps of:

s1, obtaining nucleic acid of a sample to be detected;

s2, performing reverse transcription by taking the nucleic acid sample obtained in the step S1 as a template to obtain cDNA;

s3, respectively carrying out multiplex PCR (polymerase chain reaction) amplification by using the primer set in any one of claims 1-5 by taking DNA in a sample and cDNA obtained by reverse transcription as templates, and preparing a product mixture of the DNA amplification product and the cDNA amplification product according to a certain proportion;

s4, using the product mixed solution obtained in the step S3 as a template, performing additive joint PCR amplification by using a joint sequence primer pair, and adding a sequencing joint sequence to a multiplex PCR amplified product to obtain a sequencing library;

S5, performing high-throughput sequencing on the sequencing library obtained in the step S4 to obtain a sequencing sequence;

S6, comparing the sequencing sequence with a pathogenic microorganism target sequence database, and counting and accurately comparing the number of reads of a specific pathogenic microorganism target;

s7, calculating the standardized reads number of the pathogenic microorganisms by using the following formula:

wherein, RPM is 1M reads pathogenic microorganism detection number; pathogen reads number is the number of reads detected by the pathogen; CLEAN READS number is the number of valid reads after sample preprocessing.

10. A method for detecting respiratory tract pathogenic microorganisms, comprising the steps of:

s1, obtaining nucleic acid of a sample to be detected;

s2, performing reverse transcription by taking the nucleic acid sample obtained in the step S1 as a template to obtain cDNA;

s3, respectively carrying out multiplex PCR (polymerase chain reaction) amplification by using the primer set in any one of claims 1-5 by taking DNA in a sample and cDNA obtained by reverse transcription as templates, and preparing a product mixture of the DNA amplification product and the cDNA amplification product according to a certain proportion;

s4, using the product mixed solution obtained in the step S3 as a template, performing additive joint PCR amplification by using a joint sequence primer pair, and adding a sequencing joint sequence to a multiplex PCR amplified product to obtain a sequencing library;

S5, performing high-throughput sequencing on the sequencing library obtained in the step S4 to obtain a sequencing sequence;

S6, comparing the sequencing sequence with a pathogenic microorganism target sequence database, and counting and accurately comparing the number of reads of a specific pathogenic microorganism target;

s7, calculating the standardized reads number of the pathogenic microorganisms by using the following formula:

wherein, RPM is 1M reads pathogenic microorganism detection number; pathogen reads number is the number of reads detected by the pathogen; CLEAN READS number is the number of valid reads after sample preprocessing.