CN117757962B - Kit and method for simultaneously detecting multiple pathogenic microorganisms tNGS - Google Patents
Kit and method for simultaneously detecting multiple pathogenic microorganisms tNGS Download PDFInfo
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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
Technical Field
The invention relates to the technical field of gene detection, in particular to tNGS (targeted pathogen high-throughput sequencing) kit and method for simultaneously detecting various pathogenic microorganisms.
Background
Since the 20 th century, the burden of global infectious diseases has been greatly reduced due to remarkable improvement of health conditions of foods and drinking water, rapid development of modern medical technology, and the like, due to the wide use of antibiotics, antiviral drugs and vaccines. However, infectious diseases remain one of the leading causes of morbidity and mortality worldwide.
At present, the incidence of global infectious diseases is rising, and pathogens show a trend of diversification and complicating. New infectious diseases such as coronavirus infection and H7N9 avian influenza appear successively. And the pathogens of classical infectious diseases such as HIV, multi-drug resistant mycobacterium tuberculosis, mycoplasma trachomatis and the like have dead ash reburning or new pathogenic characteristics. Various new and recurrent infectious diseases, difficult-to-find multiple infections, fever with unknown etiology and the like all bring great threat to human health.
Infectious diseases are one of the common clinical diseases, are local or systemic inflammation or organ dysfunction caused by pathogens such as bacteria, viruses, fungi, parasites and the like and products thereof, and have larger hazard and higher fatality rate. Therefore, clinical requirements for the accuracy and timeliness of the diagnosis of infectious diseases are raised.
Traditional pathogenic microorganism detection means include morphological detection, isolation culture, biochemical detection and immunological detection. These methods are limited by factors such as long cycle times, low positive rates, and high skill level requirements for the operator. The fluorescent quantitative PCR technology, the isothermal amplification technology and other molecular biological methods solve the problem of long detection period, and the detection of the specific sequence of the nucleic acid can rapidly judge the pathogen type within 2-4 hours, so that the method is suitable for simultaneously detecting fewer pathogen targets. For mixed unknown infection and infectious diseases caused by rare pathogens, means such as fluorescent quantitative PCR are plagued by the forepart.
With the development of modern genomics, high-throughput sequencing technology (also called next generation sequencing technology, NGS) has been able to directly perform high-throughput sequencing on nucleic acids in clinical samples without relying on traditional microbial culture, and then compare the nucleic acids with databases, so as to realize multiple aspects of tracing, detecting, parting, drug resistance evaluation and the like of infectious diseases, and have been receiving attention of more and more clinical and scientific researchers. Metagenomic sequencing (mNGS) used clinically at present is to perform high-throughput sequencing on total nucleic acid (DNA or RNA) in a human body sample or a specific environmental sample, and obtain relevant information of infectious pathogens by comparing databases. Since mNGS is able to detect both new and rare pathogens, it plays a difficult alternative role in the area of severe infections, particularly those caused by difficult and rare diseases: NGS was approved by the FDA in 2016 for diagnosis of pathogenic microorganisms and drug resistance virulence analysis; the 2019 pathogenic metagenome is written into the diagnosis and treatment guide of adult hospital-acquired pneumonia and ventilator-associated pneumonia; in 2019, the journal of China emergency medical science published expert consensus on the application of metagenomic diagnosis technology in critical infection.
Currently, most of the clinically applied pathogen identification sequencing technologies are metagenomic sequencing (mNGS), which can acquire all the nucleic acid sequence information in a sample, including pathogenic microorganism sequence information and human genome sequence information. The method has no preference on detection targets, and can theoretically detect all pathogenic microorganisms in a database.
However, the following technical problems exist in practical application: 1. in the metagenomic library construction process, the enzyme digestion is interrupted, namely, the non-offset interruption is carried out on all nucleic acids in the sample, wherein the non-offset interruption also comprises a large amount of human nucleic acids in the clinical sample and reagent engineering bacteria nucleic acids introduced by a library building reagent. In the sequencing data obtained later, most of the data volume is occupied by the human nucleic acid sequence, the data volume available for analysis of the actual pathogenic pathogen in the sample is relatively low, the detection sensitivity is affected, and the detection performance of the pathogen is affected by the human nucleic acid. 2. More than 90% of the sequencing data of a clinical sample is the human sequence, the actual data of the pathogen sequence occupies smaller area, and in order to further improve the pathogen detection sensitivity, the sequencing data of the sample can only be continuously improved, thus greatly increasing the detection cost.
Therefore, a new pathogen sequencing technology is urgently needed to solve the problems, eliminate the interference of human nucleic acid sequences as much as possible and improve the sensitivity and the specificity of pathogen detection without increasing the sequencing data volume.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides tNGS (targeted pathogen high-throughput sequencing) kit and method for simultaneously detecting various pathogenic microorganisms. The technical scheme of the invention is as follows:
In a first aspect, the present invention provides a primer set for detecting a pathogenic microorganism based on tNGS, 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.
Further, SEQ ID NO.1 to SEQ ID NO. 175 in the primer set is an upstream primer sequence; SEQ ID NO. 176 to SEQ ID NO. 350 are downstream primer sequences.
In a second aspect, the invention provides a kit for detecting pathogenic microorganisms based on tNGS, which is characterized by comprising the primer set.
Further, the kit is characterized by further comprising reverse transcriptase, DNA polymerase and PCR product purification magnetic beads.
Still further, 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.
Still further, the kit is characterized by further comprising a pool of multiplex PCR amplification primers, digestive enzymes, DNA ligases, sequencing adaptors and PCR reaction Mix.
In a third aspect, the present invention provides a targeted pathogen high throughput sequencing method for detection of pathogenic microorganisms for non-diagnostic purposes, characterized by the specific steps of:
Step1, extracting total DNA and RNA in a sample to be detected;
step 2, reversely transcribing the RNA obtained in the step 1 into cDNA;
Step 3, performing multiplex PCR amplification by using the total DNA obtained in the step 1 and the cDNA obtained by reverse transcription in the step 2 as templates and using an amplification primer pool;
step 4, performing enzyme treatment on the super-multiplex PCR amplification product obtained in the step 3, and digesting the amplification primer parts at two ends of the amplification product;
step 5, connecting sequencing joints at two ends of the digested PCR product obtained in the step 4;
Step 6, carrying out PCR amplification on the sequencing joint connection product obtained in the step 5 to obtain tNGS sequencing library;
Step 7, quality inspection and quantification are carried out on the tNGS sequencing library prepared in the step 6, and library mixing is carried out according to the quantitative result of the library;
Step 8, performing high-throughput sequencing on the sequencing library obtained in the step 7 after library mixing to obtain a sequencing sequence;
And 9, firstly carrying out primer recognition on the sequencing sequence, then comparing the sequencing sequence with pathogen sequences in a database, and finally determining pathogen types in a sample to be detected.
Preferably, the reaction system of the super multiplex PCR amplification is: multiPCR Mix 4. Mu.L, PCR ENHANCER. Mu.L, amplicon Primer Mix 1 5. Mu.L, total template 10. Mu.L, total reaction system 20. Mu.L; the reaction conditions for the super multiplex PCR amplification are: 95 ℃ for 2min; cycling for 30 times at 95 ℃ for 10s,60 ℃ for 30s and 72 ℃ for 30 s; the temperature was 60℃for 15min, and after the reaction was completed the product was cooled to 4 ℃.
Preferably, in the step 4, the reaction system of the amplification primer digestion is: DU Buffer 6. Mu.L and DU Enzyme 4. Mu.L, and the above product without elution of nucleic acid water was added to a total volume of 50. Mu.L; the digestion reaction conditions were: 37 ℃ for 10min;50 ℃ for 10min;65 ℃ for 5min, and the product is cooled to 4 ℃ after the reaction is finished.
Preferably, in the step 5, the reaction system for connecting the sequencing adaptors is: adapter 5. Mu.L, ligase Buffer 15. Mu. L, ligase. Mu.L, and the product of the previous step to a total volume of 80. Mu.L; the connection reaction conditions are as follows: 25 ℃ for 15min; after the reaction was completed, the product was cooled to 4 ℃.
The beneficial effects are that:
The invention is based on common multiple pathogens of different infection types, a certain number of target pathogenic microorganisms are selected by a targeted pathogenic high-throughput sequencing technology, corresponding conserved nucleic acid regions are determined by a bioinformatics method, specific primers are respectively designed for a plurality of conserved nucleic acid regions of each pathogenic microorganism, and all specific primer pairs of the target pathogenic microorganisms are combined together and mixed into an amplification primer pool.
In practical application, firstly, clinical sample nucleic acid is extracted, then the amplification primer pool is utilized to carry out multiplex PCR amplification to obtain a series of specific DNA fragments of different pathogenic microorganisms, the nucleic acid sequence information is read through a high-throughput sequencing technology, and then the specific pathogenic microorganism type information can be determined by directly comparing the specific pathogenic microorganism type information with a pathogenic conserved nucleic acid region for primer design.
Drawings
FIG. 1 is a technical roadmap for macro-gene lease sequencing (mNGS).
FIG. 2 is a graphical representation of a targeted pathogen high throughput sequencing (tNGS) technology for detecting pathogenic microorganisms according to the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate the understanding of the present invention, and the following examples are given for the purpose of illustration only and are not intended to limit the scope of the present invention. The present invention may be embodied in different forms and is not limited to the embodiments described herein. The following examples are provided for the purpose of facilitating an understanding of the disclosure of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
High throughput sequencing (NGS): is a sequencing technology capable of simultaneously reading hundreds of thousands to millions of DNA molecular sequences.
Metagenomic sequencing (mNGS): library construction and high throughput sequencing by direct extraction of nucleic acids from clinical samples, and analysis of species of pathogenic microorganism sequences contained in the samples using bioinformatics algorithms.
Targeted pathogen high throughput sequencing (tNGS): specific primers are used for carrying out targeted amplification on specific areas of target pathogenic microorganisms to obtain a large number of target DNA fragments, and then the DNA fragments are sequenced by a high-throughput sequencing technology and compared with target sequences to identify the pathogenic microorganisms.
Example 1
The kit provided by the invention is used for amplifying and enriching specific genome regions of pathogenic microorganisms in a target through ultra-multiplex PCR amplification, then obtaining specific nucleic acid sequences by using a high-throughput sequencing technology, and then comparing and parting the nucleic acid sequences by using a bioinformatics analysis technology, thereby realizing simultaneous detection of hundreds of pathogenic microorganisms.
The development process of the kit is as follows:
(1) Selection of target pathogenic microorganisms. And in the early stage, determining a target pathogenic microorganism list by searching literature and related expert consensus in the infection field.
(2) Specific primer design. And searching the related pathogen database, determining specific genome regions by software, and respectively designing primer pairs.
(3) Primer specificity confirmation. And purchasing a standard strain and a pathogenic microorganism without the standard strain for pathogenic microorganisms with standard strains or bacterial liquids to synthesize corresponding plasmids.
(4) Primer screening and replacement. Primer verification is carried out by combining PCR amplification with agarose gel electrophoresis, primers which can lead to primer dimer appearance or lower amplification efficiency are removed, and primers are redesigned and synthesized.
(5) And optimizing the proportioning condition of the primer. The amplification efficiency of different primers is high or low, and different primer proportioning concentrations are tested to balance the amplification efficiency among the primers as much as possible.
(6) And optimizing the PCR reaction conditions. Depending on the primer characteristics, an appropriate annealing temperature is selected and the number of cycles of amplification without PCR is tested.
The detection primer set of the target pathogenic microorganisms was obtained by screening through the development process described above, as shown in Table 1.
TABLE 1 target pathogenic microorganisms and corresponding tGNS primer sets therefor
Example 2
The method for detecting pathogenic microorganisms in alveolar lavage fluid of a patient with lower respiratory tract infection by using a targeted pathogenic high-throughput sequencing (tNGS) method comprises the following steps:
1. Sample pretreatment
A500. Mu.L sample of alveolar lavage fluid was taken and added to 0.2mm grinding beads and shaken on a shaker for 5min to promote cell disruption and release of nucleic acid.
2. Nucleic acid extraction
(1) To the shaken alveolar lavage fluid sample, 500. Mu.L of the nucleic acid binding solution and 20. Mu.L of proteinase K were added, and the mixture was thoroughly mixed, incubated at 50℃for 10min, and then 500. Mu.L of absolute ethanol was added for mixing.
(2) Taking the mixed liquid, adding the mixed liquid onto a centrifugal column sleeved with a collecting pipe, centrifuging at 10000rpm for 1min, and discarding the filtrate.
(3) 500. Mu.L of the rinse solution 1 was added to the column, centrifuged at 10000rpm for 1min, and the filtrate was discarded.
(4) 700. Mu.L of rinse 2 was added to the column and centrifuged at 10000rpm for 1min, and the filtrate was discarded.
(5) 500. Mu.L of rinse 2 was added to the column and centrifuged at 10000rpm for 1min, and the filtrate was discarded.
(6) The column was transferred to a new 1.5mL centrifuge tube, and 50. Mu. LNuclease-FREE WATER was suspended in the center of the column, and the column was allowed to stand at room temperature for 2min at 8000rpm for 1min to collect nucleic acid.
3. TNGS library construction
(1) The kit for targeting pathogen high-throughput sequencing (tNGS) has the composition and preservation conditions shown in the table II, and the target sequence of the pathogen microorganism in the sample to be detected is amplified and enriched by the ultra-multiplex PCR. The reaction system is as follows: multiPCR Mix 4. Mu.L, PCR ENHANCER. Mu.L, amplicon Primer Mix. Mu.L, total template 10. Mu.L, total reaction system 20. Mu.L; the reaction conditions for the super multiplex PCR are: 95 ℃ for 2min; cycling for 30 times at 95 ℃ for 10s,60 ℃ for 30s and 72 ℃ for 30 s; the temperature was 60℃for 15min, and after the reaction was completed the product was cooled to 4 ℃.
(2) Digestion is performed on the amplification primer regions at both ends of the amplification product. The reaction system is as follows: DU Buffer 6. Mu.L and DU Enzyme 4. Mu.L, and the above product without elution of nucleic acid water was added to a total volume of 50. Mu.L; the digestion reaction conditions were: 37 ℃ for 10min;50 ℃ for 10min;65 ℃ for 5min, and the product is cooled to 4 ℃ after the reaction is finished.
(3) A sequencing linker was attached. The reaction system is as follows: adapter 5. Mu.L, ligase Buffer 15. Mu. L, ligase. Mu.L, and the product of the previous step to a total volume of 80. Mu.L; the connection reaction conditions are as follows: 25 ℃ for 15min; after the reaction was completed, the product was cooled to 4 ℃. After the reaction is finished, the tNGS library for sequencing can be obtained through PCR amplification and product purification.
TABLE 2 high throughput sequencing (tNGS) kit targeting pathogens
2X RT Mix: reverse transcriptase for reverse transcription of RNA into cDNA;
MultiPCR Mix: DNA polymerase for multiplex PCR amplification;
amplicon Primer Mix 1: a pool of super multiplex PCR amplification primers;
DU Enzyme: digestive enzymes for digesting the amplification primer portions at both ends of the amplification product;
ligase: a DNA ligase for ligating a sequencing linker;
The adapter: sequencing adaptors for distinguishing a section of tagged primer sequences of different samples;
PCR Mix: a PCR reaction Mix for completing PCR amplification;
DNA clean reads: DNA purification magnetic beads are used to purify PCR amplification products.
4. Library quality inspection and quantification
(1) And (3) carrying out fragment quality inspection on the tNGS library by using an Agilent 2100 biological fragment analyzer, wherein the main fragment size of the normal library is 300-400 bp, and no obvious primer dimer stripes and large fragments exist.
(2) The dsDNA concentration of tNGS library was accurately quantified using a Qubit 4.0 fluorometer and mixed according to the quantitative results of the library.
5. Sequencing on machine
Taking 1 pmol of mixed tNGS library, preparing DNA nanospheres by using a one-step DNB preparation kit, adding corresponding reagents into a sequencing reagent tank, and performing high-throughput sequencing by using a MGISEQ-200RS gene sequencer, wherein the sequencing read length is SE50.
6. Data analysis
Firstly, performing joint identification on original off-machine data, identifying joint reads, cutting off joints and subsequent sequences, and then performing low-quality reads filtration to reserve high-quality sequencing data; and (3) carrying out primer identification on the high-quality sequencing data, comparing the primer identification with a constructed pathogenic microorganism database, and finally determining pathogen types in the sample.
7. Report interpretation
And filling in sample source information and the detected pathogen types and sequences in the samples by using a report template of a corresponding type.
Example 3
In order to verify the detection performance of the targeted pathogen high-throughput sequencing (tNGS) method provided by the invention, the same sample is subjected to metagenome sequencing and tNGS detection respectively, and the difference of the results of the metagenome sequencing and the tNGS detection is compared. For the detection of inconsistent pathogens, the verification was performed using additionally commercial fluorescent PCR kits.
The results are shown in Table 3 below:
TABLE 3 alignment of results of metagenomic sequencing (mNGS) and targeted pathogen high throughput sequencing (tNGS)
The result shows that tNGS has better detection performance, no missed detection condition occurs for the target pathogen detected by metagenome sequencing, and the additionally detected partial viruses are also verified to be true positive by fluorescence PCR.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the invention, but any modifications, equivalents, or improvements made within the principles of the present invention should be included in the scope of the 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.
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