CN117757962B - Kit and method for simultaneously detecting multiple pathogenic microorganisms tNGS - Google Patents

Abstract

The invention provides a target pathogen high-throughput sequencing kit and a method for simultaneously detecting multiple pathogen microorganisms, wherein a group of primers are obtained by designing, detecting and screening specific primers of common multiple pathogens with different infection types, a specific region of the target pathogen microorganism is subjected to target amplification by target pathogen high-throughput sequencing (tNGS), a DNA fragment of the target pathogen microorganism is obtained, and then a target sequence is compared with a pathogen sequence, so that identification of the pathogen microorganism is realized. Compared with metagenome sequencing, the targeting pathogen high-throughput sequencing can greatly reduce the data size of human nucleic acid in a tested sample, improve the detection sensitivity, increase the detection rate of pathogenic microorganisms and reduce the sample sequencing cost.

Description

A kit and method for simultaneously detecting multiple pathogenic microorganisms tNGS

Technical Field

The present invention relates to the field of gene detection technology, and more specifically to a tNGS (targeted pathogen high-throughput sequencing) kit and method for simultaneously detecting multiple pathogenic microorganisms.

Background technique

Since the 20th century, the global burden of infectious diseases has dropped significantly due to the widespread use of antibiotics, antiviral drugs and vaccines, significant improvements in food and drinking water hygiene, and the rapid development of modern medical technology. However, infectious diseases are still one of the main causes of morbidity and mortality worldwide.

At present, the incidence of infectious diseases has increased globally, and pathogens are showing a trend of diversification and complexity. New infectious diseases such as novel coronavirus infection and H7N9 avian influenza have emerged one after another. Classic infectious disease pathogens such as HIV, multidrug-resistant Mycobacterium tuberculosis, and Mycoplasma trachomatis have revived or shown new pathogenic characteristics. Various new and recurrent infectious diseases, multiple infections that are difficult to detect, and fever of unknown cause have all posed a huge threat to human health.

Infectious diseases are common clinical diseases, mostly local or systemic inflammation or organ dysfunction caused by pathogens such as bacteria, viruses, fungi and parasites and their products, which are very harmful and have a high mortality rate. Therefore, clinical diagnosis of infectious diseases places higher demands on accuracy and timeliness.

Traditional methods of pathogen detection include morphological detection, isolation and culture, biochemical detection, and immunological detection. These methods are limited by factors such as long cycles, low positive rates, and relatively high technical requirements for operators. The emergence of molecular biological methods such as fluorescent quantitative PCR technology and isothermal amplification technology has solved the problem of long detection cycles. By detecting specific nucleic acid sequences, the type of pathogen can be quickly determined within 2 to 4 hours, which is suitable for detecting fewer pathogen targets at the same time. However, for mixed unknown infections and infectious diseases caused by rare pathogens, methods such as fluorescent quantitative PCR are stretched to the limit.

With the development of modern genomics, high-throughput sequencing technology (also known as next-generation sequencing technology, NGS) has been able to do without relying on traditional microbial culture. It can directly perform high-throughput sequencing on nucleic acids in clinical samples, and then compare them with databases to achieve the traceability, detection, typing and drug resistance assessment of infectious diseases, and has attracted more and more attention from clinical and scientific researchers. Metagenomic sequencing (mNGS) currently used in clinical practice is a high-throughput sequencing of total nucleic acids (DNA or RNA) in human samples or specific environmental samples, and obtains relevant information about infectious pathogens by comparing databases. Because mNGS can detect emerging and rare pathogens, it plays an irreplaceable role in the field of severe infections, especially severe infections caused by difficult and rare diseases: in 2016, the FDA approved NGS for the diagnosis and drug resistance virulence analysis of pathogenic microorganisms; in 2019, pathogenic metagenomes were written into the diagnosis and treatment guidelines for adult hospital-acquired pneumonia and ventilator-associated pneumonia; in 2019, the Chinese Journal of Emergency Medicine published the "Expert Consensus on the Application of Metagenomic Diagnostic Technology in Critical Infections".

The most widely used pathogen identification sequencing technology in clinical practice is metagenomic sequencing (mNGS), which can obtain all nucleic acid sequence information in a sample, including pathogen sequence information and human genome sequence information. This method has no preference for the detection target and can theoretically detect all pathogens in the database.

However, the following technical problems still exist in practical applications: 1. In the process of metagenomic library construction, the enzyme cutting and interruption step is to interrupt all nucleic acids in the sample without bias, including a large amount of human nucleic acids in clinical samples and reagent-engineered bacterial nucleic acids introduced by library construction reagents. In the subsequent sequencing data, the vast majority of the data is also occupied by human nucleic acid sequences. The amount of data available for analysis of actual pathogens in the sample is relatively low, and the detection sensitivity will be affected. Therefore, human nucleic acids will affect the detection performance of pathogens. 2. In the sequencing data of a clinical sample, more than 90% of the data is human sequences, and the actual amount of pathogen sequences is relatively small. In order to further improve the sensitivity of pathogen detection in clinical practice, the amount of sequencing data of the sample can only be continuously increased, which also greatly increases the detection cost.

Therefore, a new pathogen sequencing technology is urgently needed to solve these problems. Without increasing the amount of sequencing data, it is necessary to eliminate the interference of human nucleic acid sequences as much as possible and improve the sensitivity and specificity of pathogen detection.

Summary of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to propose a tNGS (targeted pathogen high-throughput sequencing) kit and method for simultaneously detecting multiple pathogenic microorganisms. The technical solution of the present invention is as follows:

In a first aspect, the present invention provides a primer set for pathogenic microorganism detection based on tNGS, characterized in that the primer set specifically captures the coding region sequences of the following pathogenic microorganism genomes: Acinetobacter baumannii, Bordetella pertussis, Burkholderia cepacia, Chlamydia pneumoniae, Chlamydia psittaci, Corynebacterium diphtheriae, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Haemophilus influenzae, Klebsiella aerogenes, Klebsiella pneumoniae, Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium tuberculosis complex, Mycobacterium avium, Mycobacterium intracellulare Bacillus, Mycobacterium kansasii, Mycobacterium massiliense, Mycobacterium tuberculosis, Mycobacterium abscessus, Mycobacterium chelonae, Mycoplasma pneumoniae, Neisseria meningitidis, Nocardia brasiliensis, Nocardia gelsenkirchen, Nocardia dermatophytes, Nocardia novae, Nocardia otitis media, Orientia tsutsugamushi, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus ridge, Streptococcus pneumoniae, Streptococcus pyogenes, BK polyomavirus, Hepatitis B virus, Human adenovirus type D, Herpes simplex virus type 1, Herpes simplex virus type 2, Varicella-zoster virus, human cytomegalovirus, human herpesvirus-7, human bocavirus-1, Epstein-Barr virus, human herpesvirus-6, human adenovirus B, human adenovirus C, human adenovirus E, human parvovirus B19, JC polyomavirus, KI polyomavirus, WU polyomavirus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, Cryptococcus neoformans, Fusarium spp., Pneumocystis jiroveci, Rhizomucor microsporus, Rhizopus oryzae, Scedosporium apiospermum, Talaromyces marneffei, human coronavirus-22 9E, human coronavirus HKU1, human coronavirus NL63, human coronavirus OC43, human metapneumovirus, human respiratory syncytial virus B, influenza A virus H1N1, influenza A virus H2N2, influenza A virus H3N2, influenza A virus H5N1, influenza A virus H7N9, influenza A virus H9N2, influenza B virus, Japanese encephalitis virus, Middle East respiratory syndrome virus, human respiratory syncytial virus A, human rhinovirus A, human rhinovirus B, human rhinovirus C, new coronavirus, SARS coronavirus;

The primer set comprises primers with sequences shown as SEQ ID NO: 1 to SEQ ID NO: 350.

Furthermore, in the primer set, SEQ ID NO: 1 to SEQ ID NO: 175 are upstream primer sequences; SEQ ID NO: 176 to SEQ ID NO: 350 are downstream primer sequences.

In a second aspect, the present invention provides a kit for detecting pathogenic microorganisms based on tNGS, characterized in that it comprises the above-mentioned primer set.

Furthermore, the kit is characterized in that it also includes reverse transcriptase, DNA polymerase and PCR product purification magnetic beads.

Furthermore, the kit is characterized in that the reverse transcriptase is 2x RT Mix, the DNA polymerase is Multi-PCR Mix, and the PCR product purification magnetic beads are DNA clean beads.

Furthermore, the kit is characterized in that it also includes a super-multiplex PCR amplification primer pool, a digestion enzyme, a DNA ligase, a sequencing adapter and a PCR reaction mix.

In a third aspect, the present invention provides a targeted pathogen high-throughput sequencing method for detecting pathogenic microorganisms for non-diagnostic purposes, characterized in that it specifically comprises the following steps:

Step 1, extracting total DNA and RNA from the sample to be tested;

Step 2, reverse transcribing the RNA obtained in step 1 into cDNA;

Step 3, using the total DNA obtained in step 1 and the cDNA obtained by reverse transcription in step 2 as templates, and performing super-multiplex PCR amplification using an amplification primer pool;

Step 4, treating the super multiplex PCR amplification product obtained in step 3 with an enzyme to digest the amplification primers at both ends of the amplification product;

Step 5, connecting the two ends of the digested PCR product obtained in step 4 to sequencing adapters;

Step 6, performing PCR amplification on the sequencing adapter ligation product obtained in step 5 to obtain a tNGS sequencing library;

Step 7, quality inspection and quantification of the tNGS sequencing library prepared in step 6, and library mixing based on the library quantification results;

Step 8, performing high-throughput sequencing on the mixed sequencing library obtained in step 7 to obtain a sequencing sequence;

Step 9: First perform primer identification on the sequencing sequence, then compare it with the pathogen sequence in the database, and finally determine the type of pathogen in the sample to be tested.

Preferably, the reaction system of the super-multiplex PCR amplification is: Multi-PCR Mix 4μL, PCR Enhancer 1μL, Amplicon Primer Mix 1 5μL, total template 10μL, and the total reaction system is 20μL; the reaction conditions of the super-multiplex PCR amplification are: 95℃2min; 95℃10s, 60℃30s, 72℃30s, 30 cycles; 60℃15min, and the product is cooled to 4℃ after the reaction is completed.

Preferably, in step 4, the reaction system for digestion of the amplification primer is: 6 μL of DU Buffer, 4 μL of DU Enzyme, and the product of the previous step eluted with nucleic acid-free water is added to a total volume of 50 μL; the digestion reaction conditions are: 37°C for 10 min; 50°C for 10 min; 65°C for 5 min, and the product is cooled to 4°C after the reaction is completed.

Preferably, in step 5, the reaction system for connecting the sequencing adapter is: Adaptor 5 μL, Ligase Buffer 15 μL, Ligase 10 μL, and the product of the previous step to a total volume of 80 μL; the connection reaction conditions are: 25°C for 15 min; after the reaction is completed, the product is cooled to 4°C.

Beneficial effects:

The present invention is based on common common pathogens of different infection types. The targeted pathogen high-throughput sequencing technology first selects a certain number of target pathogenic microorganisms, determines their corresponding conserved nucleic acid regions through bioinformatics methods, designs specific primers for multiple conserved nucleic acid regions of each pathogenic microorganism, and then merges all specific primer pairs of these target pathogenic microorganisms together to mix into an amplification primer pool.

In actual application, the nucleic acid of the clinical sample is first extracted, and then the amplification primer pool is used to perform super-multiplex PCR amplification to obtain a series of specific DNA fragments of different pathogenic microorganisms. The nucleic acid sequence information is read by high-throughput sequencing technology, and then directly compared with the conserved nucleic acid region of the pathogen used for primer design to determine the type information of the specific pathogenic microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the technical roadmap of metagenomic sequencing (mNGS).

FIG2 is a technical roadmap of targeted pathogen high-throughput sequencing (tNGS) for detecting pathogenic microorganisms according to the present invention.

Detailed ways

For ease of understanding of the present invention, the present invention will be described more fully below, and the following examples are only used to specifically illustrate the present invention, and are not intended to limit the scope of the present invention. The present invention can be implemented in different forms and is not limited to the embodiments described herein. The purpose of providing the following examples is only to facilitate understanding of the disclosure of the present invention.

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

High-throughput sequencing (NGS): is a sequencing technology that can read hundreds of thousands to millions of DNA molecule sequences simultaneously.

Metagenomic sequencing (mNGS): By directly extracting nucleic acids from clinical samples for library construction and high-throughput sequencing, bioinformatics algorithms are used to analyze the types of pathogenic microbial sequences contained in the samples.

Targeted pathogen high-throughput sequencing (tNGS): Use specific primers to perform targeted amplification on specific regions of target pathogenic microorganisms to obtain a large number of target DNA fragments. These DNA fragments are then sequenced using high-throughput sequencing technology and compared with the target sequence to identify the pathogenic microorganism.

Example 1

The kit described in the present invention is planned to amplify and enrich the specific genomic regions of the target pathogenic microorganisms through super-multiplex PCR amplification, then use high-throughput sequencing technology to obtain specific nucleic acid sequences, and then use bioinformatics analysis technology to compare and type these nucleic acid sequences, thereby realizing the simultaneous detection of hundreds of pathogenic microorganisms.

The kit development process is as follows:

(1) Selection of target pathogenic microorganisms. In the early stage, the list of target pathogenic microorganisms was determined by searching the literature and consensus of experts in the field of infection.

(2) Specific primer design: Search the relevant pathogen database, determine the specific genomic region through software, and design primer pairs respectively.

(3) Confirmation of primer specificity: For pathogenic microorganisms with standard strains or bacterial liquids, purchase standard strains; for pathogenic microorganisms without standard strains, synthesize corresponding plasmids.

(4) Primer screening and replacement: Primers were verified by PCR amplification combined with agarose gel electrophoresis, and primers that would cause primer dimers or have low amplification efficiency were removed. Primers were then redesigned and synthesized.

(5) Optimization of primer ratio conditions. The amplification efficiency of different primers varies. In order to balance the amplification efficiency of each primer as much as possible, different primer ratio concentrations were tested.

(6) Optimize PCR reaction conditions. Select the appropriate annealing temperature based on the characteristics of the primers and test different PCR amplification cycle numbers.

Through the above development process, a detection primer set for the target pathogenic hospital microorganisms was screened and obtained, as shown in Table 1.

Table 1 Target pathogenic microorganisms and their corresponding tGNS primer sets

Example 2

The targeted pathogen high-throughput sequencing (tNGS) method is used to detect pathogenic microorganisms in the bronchoalveolar lavage fluid of patients with lower respiratory tract infection. The entire detection process is as follows:

1. Sample pretreatment

Take 500 μL of bronchoalveolar lavage fluid sample, add 0.2 mm grinding beads, and oscillate on an oscillator for 5 minutes to break the cells and release nucleic acids.

2. Nucleic acid extraction

(1) Add 500 μL of nucleic acid binding solution and 20 μL of proteinase K to the shaken bronchoalveolar lavage fluid sample, mix thoroughly, incubate at 50°C for 10 min, and then add 500 μL of anhydrous ethanol to mix.

(2) Take the mixed liquid and add it to a centrifuge column with a collection tube. Centrifuge at 10,000 rpm for 1 min and discard the filtrate.

(3) Add 500 μL of rinse solution 1 to the centrifuge column, centrifuge at 10,000 rpm for 1 min, and discard the filtrate.

(4) Add 700 μL of rinse solution 2 to the centrifuge column, centrifuge at 10,000 rpm for 1 min, and discard the filtrate.

(5) Add 500 μL of rinse solution 2 to the centrifuge column, centrifuge at 10,000 rpm for 1 min, and discard the filtrate.

(6) Transfer the centrifuge column to a new 1.5 mL centrifuge tube, add 50 μL of Nuclease-Free Water to the center of the centrifuge column, let it stand at room temperature for 2 min, and centrifuge at 8000 rpm for 1 min to collect the nucleic acid.

3. tNGS library construction

(1) Using the targeted pathogen high-throughput sequencing (tNGS) kit of the present invention, the kit composition and storage conditions are shown in Table 2, firstly, the pathogenic microorganism nucleic acid target sequence in the sample to be tested is enriched by super-multiplex PCR amplification. The reaction system is: Multi-PCR Mix 4μL, PCR Enhancer 1μL, Amplicon Primer Mix 15μL, total template 10μL, total reaction system 20μL; the reaction conditions of super-multiplex PCR are: 95℃2min; 95℃10s, 60℃30s, 72℃30s, 30 cycles; 60℃15min, after the reaction, the product is cooled to 4℃.

(2) Digest the amplification primer regions at both ends of the amplification product. The reaction system is: 6μL DU Buffer, 4μL DU Enzyme, add the product of the previous step eluted with nuclease-free water to a total volume of 50μL; the digestion reaction conditions are: 37℃10min; 50℃10min; 65℃5min, and the product is cooled to 4℃ after the reaction.

(3) Connect the sequencing adapter. The reaction system is: Adaptor 5μL, Ligase Buffer 15μL, Ligase 10μL, and the product of the previous step to a total volume of 80μL; the connection reaction conditions are: 25℃15min; after the reaction, the product is cooled to 4℃. After the reaction, PCR amplification and product purification are performed once more to obtain the tNGS library that can be sequenced.

Table 2 Targeted pathogen high-throughput sequencing (tNGS) kits

2x RT Mix: Reverse transcriptase, used to reverse transcribe RNA into cDNA;

Multi-PCR Mix: DNA polymerase for multiplex PCR amplification;

Amplicon Primer Mix 1: Super multiplex PCR amplification primer pool;

DU Enzyme: digestive enzyme, used to digest the amplification primer parts at both ends of the amplification product;

Ligase: DNA ligase, used to connect sequencing adapters;

Adaptor: Sequencing adapter, a labeled primer sequence used to distinguish different samples;

PCR Mix: PCR reaction Mix, used to complete PCR amplification;

DNA clean beads: DNA purification magnetic beads, used to purify PCR amplification products.

4. Library quality control and quantification

(1) The tNGS library was quality checked using the Agilent 2100 Bio-Fragment Analyzer. The main fragment size of a normal library was between 300 bp and 400 bp, and there were no obvious primer dimer bands or large fragments.

(2) Use the Qubit 4.0 fluorometer to accurately quantify the dsDNA concentration of the tNGS library and mix the library according to the quantitative results.

5. Sequencing

Take 1 p mol of the mixed tNGS library, use the one-step DNB preparation kit to prepare DNA nanoballs, add the corresponding reagents to the sequencing reagent tank, and use the MGISEQ-200RS gene sequencer for high-throughput sequencing with a sequencing read length of SE50.

6. Data Analysis

The original offline data is first subjected to connector recognition, connector reads are identified, connectors and subsequent sequences are truncated, and low-quality reads are filtered to retain high-quality sequencing data. Primers are identified for high-quality sequencing data, and then compared with the constructed pathogenic microorganism database to finally determine the type of pathogen in the sample.

7. Report Interpretation

Use the corresponding type of report template to fill in the sample source information and the types and sequence numbers of pathogens detected in the sample.

Example 3

In order to verify the detection performance of the targeted pathogen high-throughput sequencing (tNGS) method provided by the present invention, metagenomic sequencing and tNGS detection were performed on the same sample, and the differences between the two results were compared. For pathogens with inconsistent detection results, a commercial fluorescent PCR kit was used for additional verification.

The results are shown in Table 3 below:

Table 3 Comparison of metagenomic sequencing (mNGS) and targeted pathogen high-throughput sequencing (tNGS) results

The results showed that tNGS had good detection performance, and there were no missed detections of target pathogens detected by metagenomic sequencing. In addition, some of the additional viruses detected were also verified as true positives by fluorescent PCR.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the principle of the present invention should be included in the protection scope of the present invention.

Claims (5)

1. A primer set for tNGS-based detection of a pathogenic microorganism, wherein the primer set specifically captures the sequence of the genomic coding region of the pathogenic microorganism: acinetobacter baumannii, bordetella pertussis, burkholderia cepacia, chlamydia pneumoniae, chlamydia psittaci, diphtheria bacillus, enterobacter cloacae, enterococcus faecium, escherichia coli, haemophilus influenzae, klebsiella aerogenes, klebsiella pneumoniae, legionella pneumophila, listeria monocytogenes, moraxella catarrhalis, mycobacterium tuberculosis complex, mycobacterium avium, mycobacterium intracellulare, mycobacterium kansasii, mycobacterium mosaic, mycobacterium tuberculosis, mycobacterium abscess, mycobacterium terrae, mycoplasma pneumoniae, neisseria meningitidis, pasteurella, georgi nocardia, nanocardia piricola, nanocardia gangrenorphis, nanocardia vensis, nakava, orientia tsutsugamushi, proteus, pseudomonas aeruginosa, salmonella, serratia, staphylococcus aureus, M. maltophilia, streptococcus cristatus, streptococcus pneumoniae, streptococcus pyogenes, BK polyoma virus, hepatitis B virus, human adenovirus type D, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, human cytomegalovirus, human herpes virus type 7, human Bokavirus type 1, epstein-Barr virus, human herpesvirus type 6, human adenovirus type B, human adenovirus type C, human adenovirus type E, human parvovirus B19, JC polyomavirus, KI polyomavirus, WU polyomavirus, aspergillus flavus, aspergillus fumigatus, aspergillus niger, aspergillus terreus, candida albicans, candida glabrata, candida krusei, candida tropicalis, novel cryptococcus, fusarium, yersinia pneumosporum, rhizomucor minutissimi, rhizopus, saccharomyces cuspidatus, nimarxianus, human coronavirus type 229E, human coronavirus HKU1, human coronavirus NL63, human coronavirus OC43, human metapneumovirus, human respiratory syncytial virus B, influenza a virus H1N1, influenza a virus H2N2, influenza a virus H3N2, influenza a virus H5N1, influenza a virus H7N9, influenza a virus H9N2, influenza B virus, japanese encephalitis B virus, middle eastern respiratory syndrome virus, human respiratory syncytial virus a, human rhinovirus B, human rhinovirus C, novel coronavirus, SARS coronavirus; the primer group comprises primers with sequences shown as SEQ ID NO. 1 to SEQ ID NO. 350.

2. The primer set of claim 1, wherein SEQ ID NO. 1 through SEQ ID NO. 175 are upstream primer sequences; SEQ ID NO. 176 to SEQ ID NO. 350 are downstream primer sequences.

3. A kit for tNGS-based detection of a pathogenic microorganism comprising the primer set of claim 1 or claim 2.

4. The kit of claim 3, further comprising reverse transcriptase, DNA polymerase and PCR product purification magnetic beads.

5. The kit of claim 4, wherein the reverse transcriptase is 2x RT Mix, the DNA polymerase is Multi-PCR Mix, and the PCR product purification beads are DNA clean beads.