Tropical pathogen spectrum multiple detection primer group based on tNGS technology and application of tropical pathogen spectrum multiple detection primer group

Abstract

The invention discloses a tropical pathogen spectrum multiple detection primer group based on a tNGS technology and application thereof. The multiple detection primer group comprises primers as shown in SEQ NO.: 1 to SEQ NO.: 260 in a sequence table. By utilizing the multiple detection primer group, 100 common pathogens in the tropical region can be efficiently and accurately detected in the same reaction system.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection, and particularly to a multiplex detection primer set for tropical pathogen spectra based on tNGS technology and its application. Background Art

[0002] The development of pathogen detection technology has evolved from traditional morphological and biochemical detection to modern molecular biology detection. With the rapid development of gene sequencing technology, high-throughput sequencing technology (NGS) has gradually become an important tool for pathogen detection. However, although traditional metagenomic sequencing (mNGS) can detect multiple pathogens, it is difficult to meet the needs of rapid clinical detection due to high sequencing costs, complex data analysis, and the need for a large sample size. In recent years, the emergence of tNGS technology has provided a new solution for pathogen detection. tNGS captures and sequences target regions by designing specific primer pairs, significantly improving the sensitivity and specificity of detection. However, existing tNGS technologies still have problems such as limited detection range, insufficient sensitivity, and complex detection processes when detecting multiple pathogens. Summary of the Invention

[0003] Aiming at the deficiencies of the above-mentioned prior art, the present invention proposes a multiplex detection primer set for tropical pathogen spectra based on tNGS technology and its application.

[0004] The multiplex detection primer set for tropical pathogen spectra provided by the present invention includes the primers shown in SEQ NO.: 1 to SEQ NO.: 260 in the sequence listing.

[0005] In one embodiment, the primer set targets the genomes of 100 pathogenic microorganisms in the tropical region, 4 drug resistance genes, and 4 Mycoplasma pneumoniae mutation sites.

[0006] In one embodiment, the 100 pathogenic microorganisms in the tropical region are influenza A virus, influenza B virus, Clostridium perfringens, human bocavirus 1, human immunodeficiency virus type 2, human coronavirus 229E, human herpesvirus 3, human herpesvirus 5, human adenovirus group A, Borrelia burgdorferi, Trypanosoma cruzi, Crimean-Congo hemorrhagic fever virus, Fusobacterium nucleatum, Toxoplasma gondii, Vibrio vulnificus, Leishmania, Haemophilus parainfluenzae, Listeria monocytogenes, respiratory syncytial virus A, Legionella pneumophila, Stenotrophomonas maltophilia, Francisella tularensis, Chikungunya virus, Echinococcus multilocularis, Escherichia coli, Zika virus, Fusarium oxysporum complex, Trypanosoma brucei, Brucella, Angiostrongylus cantonensis, La Crosse virus, novel bunyavirus, Cryptococcus neoformans, Coxsackievirus A16, Rhizopus, Acanthamoeba, Trichomonas, Mucorales, Hantavirus, Bartonella henselae, Salmonella, Powassan virus, Burkholderia cepacia, Entamoeba histolytica, Bacillus anthracis, Taenia solium, Monkeypox virus, Plasmodium, Dengue virus, Candida albicans, Bordetella pertussis, Rickettsia, Burkholderia pseudomallei, Strongyloides stercoralis, Enterococcus faecalis, Ureaplasma parvum, Echinococcus granulosus, Mycobacterium tuberculosis complex, Pneumocystis jirovecii, Enterovirus, Paragonimus, Klebsiella pneumoniae, Chlamydia pneumoniae, Streptococcus pneumoniae, Bacteroides fragilis, Elizabethkingia meningoseptica, Treponema pallidum, Histoplasma capsulatum, Tick-borne encephalitis virus, West Nile encephalitis virus, Nocardia, Norovirus, Rotavirus, Staphylococcus aureus, Leptospira, Pseudomonas aeruginosa, Enterobacter cloacae complex, Cryptosporidium, Vibrio cholerae, Talaromyces marneffei, Acinetobacter baumannii, Measles virus, Aspergillus flavus complex, Yellow fever virus, Yersinia pestis, Rhinovirus, Mycobacterium xanthus, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium ulcerans, Mycobacterium scrofulaceum, Mycobacterium malmoense, Mycobacterium vaccae, Mycobacterium avium-intracellulare complex, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium haemophilum, Mycobacterium simiae, Mycobacterium leprae.

[0007] In one embodiment, the 4 drug resistance genes are blaCTX-M, mecA, mecC, mecB.

[0008] In one embodiment, the 4 Mycoplasma pneumoniae mutation sites are the 2063-site mutation, the 2064-site mutation, the 2067-site mutation, and the 2617-site mutation.

[0009] The multiplex detection kit for tropical pathogen spectrum provided by the present invention contains the primer group described above.

[0010] In one embodiment, the kit further comprises one or more of reverse transcriptase, DNA polymerase, DNA purification magnetic beads, nuclease-free water, absolute ethanol, and Qubit quantification reagent.

[0011] The multiplex detection method for tropical pathogen spectrum based on tNGS technology provided by the present invention includes: Extracting the nucleic acid of the sample to be tested as a template; Performing multiplex PCR amplification using the primer group described above; Constructing a tNGS sequencing library using the multiplex PCR amplification product; Performing high-throughput sequencing on the sequencing library; Performing bioinformatics analysis and comparison on the sequencing results to determine the types of pathogens in the sample to be tested.

[0012] In one embodiment, when the nucleic acid is RNA, reverse transcription is used to transcribe the RNA into cDNA.

[0013] In one embodiment, the multiplex PCR amplification product also undergoes one or both of purification and a second round of multiplex PCR amplification.

[0014] In one embodiment, the high-throughput sequencing platform includes the MGI sequencing platform or the Illumina sequencing platform or other sequencing platforms with the same sequencing principle.

[0015] The multiplex detection primers for tropical pathogen spectrum based on tNGS technology proposed by the present invention target the target sequences of epidemic pathogens in the tropical region by designing specific primers for gene fragments of common pathogenic microorganisms in the tropical region, and can efficiently and accurately detect 100 common pathogens in the tropical region in the same reaction system. Detailed implementation mode

[0016] Definition: tNGS (targeted Next-Generation Sequencing): Targeted region high-throughput sequencing, which is a technology for sequencing specific gene regions, featuring high throughput, high sensitivity, and high specificity.

[0017] To make the technical solution of the present invention clearer, the following further elaborates on the present invention with specific embodiments.

[0018] Example 1

[0019] Determine the scope of common pathogenic microorganisms in the tropical region. Based on the real epidemiological data in the tropical region, and by searching the literature and relevant expert consensus in the field of infection, determine the scope of common pathogenic microorganisms in the tropical region. Finally, 100 pathogenic microorganisms in the tropical region, as well as the detection of 4 drug resistance genes and 4 Mycoplasma pneumoniae mutation sites, are determined.

[0020] The 100 pathogenic microorganisms in the tropical region are: Influenza A virus, Influenza B virus, Clostridium perfringens, Human bocavirus 1, Human immunodeficiency virus 2, Human coronavirus 229E, Human herpesvirus 3, Human herpesvirus 5, Human adenovirus group A, Borrelia burgdorferi, Trypanosoma cruzi, Crimean-Congo hemorrhagic fever virus, Fusobacterium nucleatum, Toxoplasma gondii, Vibrio vulnificus, Leishmania, Haemophilus parainfluenzae, Listeria monocytogenes, Respiratory syncytial virus A, Legionella pneumophila, Stenotrophomonas maltophilia, Francisella tularensis, Chikungunya virus, Echinococcus multilocularis, Escherichia coli, Zika virus, Fusarium oxysporum complex, Trypanosoma brucei, Brucella, Angiostrongylus cantonensis, La Crosse virus, Novel bunyavirus, Cryptococcus neoformans, Coxsackievirus A16, Rhizopus, Acanthamoeba, Trichomonas, Mucorales, Hantavirus, Bartonella henselae, Salmonella, Powassan virus, Burkholderia cepacia, Entamoeba histolytica, Bacillus anthracis, Taenia solium, Monkeypox virus, Plasmodium, Dengue virus, Candida albicans, Bordetella pertussis, Rickettsia, Burkholderia pseudomallei, Strongyloides stercoralis, Enterococcus faecalis, Ureaplasma parvum, Echinococcus granulosus, Mycobacterium tuberculosis complex, Pneumocystis jirovecii, Enterovirus, Paragonimus, Klebsiella pneumoniae, Chlamydia pneumoniae, Streptococcus pneumoniae, Bacteroides fragilis, Elizabethkingia meningoseptica, Treponema pallidum, Histoplasma capsulatum, Tick-borne encephalitis virus, West Nile encephalitis virus, Nocardia, Norovirus, Rotavirus, Staphylococcus aureus, Leptospira, Pseudomonas aeruginosa, Enterobacter cloacae complex, Cryptosporidium, Vibrio cholerae, Talaromyces marneffei, Acinetobacter baumannii, Measles virus, Aspergillus flavus complex, Yellow fever virus, Yersinia pestis, Rhinovirus, Mycobacterium xanthus, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium ulcerans, Mycobacterium scrofulaceum, Mycobacterium malmoense, Mycobacterium bovis, Mycobacterium avium-intracellulare complex, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium haemophilum, Mycobacterium simiae, Mycobacterium leprae.

[0021] The 4 drug resistance genes are blaCTX-M, mecA, mecC, mecB.

[0022] The 4 Mycoplasma pneumoniae mutation sites are point mutations at 2063 / 2064 / 2067 / 2617.

[0023] Specific primer design. Download high-quality pathogenic microorganism genomes, construct a local genome database, determine specific genomic sequences through software, and design primer pairs. The primer length range is set at 18 - 25 bp, and the GC content is controlled at 40% - 60%, avoiding more than 3 consecutive identical bases.

[0024] Specific primer screening. Purchase standard strains, verify the primer performance, and remove primers that cause primer dimers or have low amplification efficiency.

[0025] Through the above development process, a primer set for detecting the target pathogenic microorganism is screened and obtained, as shown in Table 1.

[0026] Table 1. Target pathogenic microorganisms provided by the present invention and their corresponding tNGS primer set sequences

[0027]

[0028]

[0029]

[0030]

[0031]

[0032] Use the above primer set to detect patient samples, including the following steps: Gently shake and mix the sample, aspirate 800 μL of the corresponding sample, add 80 μL of lysis buffer Buffer L, and transfer it to a pre-loaded grinding tube. Place the grinding tube containing the sample into a cell disruptor, run the cell disruption program (vibration intensity of 6 M / S, grinding time of 30 s, interval time of 30 s, 6 cycles), and centrifuge at 12,000 rpm for 3 min after cell disruption to eliminate foam; transfer 600 μL of the supernatant to well A of a deep well plate; finally, add 40 μL of proteinase Proteinase K to well A of the deep well plate; insert a stirring sleeve into row B of the deep well plate and place it on the workbench of an automated extractor; start the pre-set program, turn on the automated extraction, and run for about 30 min; after the program ends, take out the deep well plate, transfer the nucleic acid in well F to a 1.5 mL centrifuge tube and measure the Qubit concentration.

[0033] Take a 0.2 mL PCR tube and prepare the reaction system in the tube according to Table 2 below.

[0034] Table 2 Multiplex PCR reaction system

[0035] After the above system is prepared, vortex and mix well, centrifuge instantaneously, place it on a PCR instrument, and perform the reaction according to the program in Table 3 below.

[0036] Table 3 PCR amplification system

[0037] Before use, the DNA purification magnetic beads were equilibrated at room temperature for 30 min and mixed well. 45 µL of magnetic beads were added to the above PCR products. After shaking and mixing well, the mixture was left standing at room temperature for 5 min, centrifuged briefly, placed on a magnetic stand and left standing for 3 min, and the supernatant was discarded. 50 μL of Cleanbuffer was added, gently shaken and mixed well, left standing at 37 °C in a PCR instrument for 5 min, centrifuged briefly, placed on a magnetic stand and left standing for 3 min, and the supernatant was discarded. After discarding, it was further discarded cleanly with a 10-µL pipette. 200 µL of 80% ethanol was added, left standing for 30 s, and the supernatant was discarded. This was repeated once. The centrifuge tube was placed on a magnetic stand, the excess liquid was aspirated off, and it was air-dried at room temperature. 30 µL of nuclease-free water was added for elution. After shaking and mixing well, it was left standing at room temperature for 5 min, centrifuged briefly and then placed on a magnetic stand and left standing for 3 min, and 28 µL of the supernatant was aspirated into a new centrifuge tube (the product was the tNGS library). 1 µL was taken and the concentration was measured and recorded using Qubit.

[0038] 1 pmol of the mixed tNGS library was taken, and a one-step DNB preparation kit was used to prepare DNA nanospheres. The corresponding reagents were added to the sequencing reagent tank, and a MGISEQ-20S gene sequencer was used for high-throughput sequencing with a sequencing read length of SE50. The original off-machine data was first quality-controlled and adapter sequences were removed, and bioinformatics analysis procedures were used to analyze the cleaned data to obtain the detection results of pathogenic microorganisms.

[0039] In order to verify the detection performance of the primer set provided by the present invention, targeted pathogen high-throughput sequencing (tNGS) detection was performed on known pathogen positive samples, and a commercially available certified PCR product was used as a control for detection to compare the differences in the results between the two. The results are shown in Table 4 below.

[0040] Table 4. Result comparison

[0041] The results showed that the tNGS had excellent detection performance, no missed detections occurred, and the results were consistent with those of fluorescence PCR detection.

[0042] Based on the real epidemiological data in tropical regions, the present invention selected 100 tropical pathogenic microorganisms, covering regionally highly threatening pathogens with high incidence in tropical regions (such as malaria, dengue fever, Mycobacterium tuberculosis, etc.), and solved the problem of missed detections caused by regional limitations of traditional detection methods. Through the combined analysis of 4 drug resistance genes that are of greatest concern clinically and 4 mutation sites of Mycoplasma pneumoniae, a precise medication plan of "one drug, one strategy" can be provided for patients, reducing the risk of abuse of broad-spectrum antibiotics.

[0043] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A multiplex detection primer set for tropical pathogens, characterized in that: The primer set includes primers shown in SEQ NO.: 1 to SEQ NO.: 260 in the sequence table.

2. The primer set according to claim 1, characterized in that The primer set targets the genomes of 100 tropical pathogenic microorganisms, 4 drug-resistant genes, and 4 Mycoplasma pneumoniae mutation sites.

3. The primer set according to claim 2, characterized in that The 100 tropical pathogenic microorganisms are influenza A virus, influenza B virus, Clostridium perfringens, human bocavirus type 1, human immunodeficiency virus type 2, human coronavirus 229E, human herpesvirus type 3, human herpesvirus type 5, human adenovirus group A, Borrelia burgdorferi, Trypanosoma cruzi, Crimean-Congo hemorrhagic fever virus, Fusobacterium nucleatum, Toxoplasma gondii, Vibrio vulnificus, Leishmania, Haemophilus parainfluenzae, Listeria monocytogenes, respiratory syncytial virus A, Legionella pneumophila , Stenotrophomonas maltophilia, Francisella tularensis, Chikungunya virus, Echinococcus multilocularis, Escherichia coli, Zika virus, Fusarium oxysporum complex, Trypanosoma brucei, Brucella, Angiostrongylus cantonensis, La Crosse virus, Novel Bunyavirus, Cryptococcus neoformans, Coxsackievirus A16, Rhizopus, Acanthamoeba, Trichomonas, Mucorales, Hantavirus, Bartonella henselae, Salmonella, Powassan virus, Burkholderia cepacia, Entamoeba histolytica, Bacillus anthracis, Taenia solium, Monkeypox virus, Plasmodium, dengue virus, Candida albicans, Bordetella pertussis, Rickettsia, Burkholderia pseudomallei, Strongyloides stercoralis, Enterococcus faecalis, Ureaplasma parvum, Echinococcus granulosus, Mycobacterium tuberculosis complex, Pneumocystis jiroveci, enterovirus, Paragonimus, Klebsiella pneumoniae, Chlamydia pneumoniae, Streptococcus pneumoniae, Bacteroides fragilis, Elizabethella meningoseptica, Treponema pallidum, Histoplasma capsulatum, tick-borne encephalitis virus, West Nile encephalitis virus, Nocardia, Norovirus, Rotavirus, Staphylococcus aureus Bacteria, Leptospira, Pseudomonas aeruginosa, Enterobacter cloacae complex, Cryptosporidium, Vibrio cholerae, Talaromyces marneffei, Acinetobacter baumannii, measles virus, Aspergillus flavus complex, yellow fever virus, Yersinia pestis, rhinovirus, Mycobacterium tomentosa, Mycobacterium gordonii, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium ulcerans, Mycobacterium scrofula, Mycobacterium malmoe, Mycobacterium vaccae, Mycobacterium avium-intracellulare complex, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium hemophilus, Mycobacterium simianum, and Mycobacterium leprae.

4. The primer set according to claim 2, characterized in that The four drug-resistant genes are blaCTX-M, mecA, mecC, and mecB.

5. The primer set according to claim 2, characterized in that: The four Mycoplasma pneumoniae mutation sites are site 2063 mutation, site 2064 mutation, site 2067 mutation, and site 2617 mutation.

6. A tropical pathogen spectrum multiplex detection kit, comprising the primer set according to any one of claims 1 to 5.

7. The kit according to claim 6, characterized in that The kit further comprises one or more of reverse transcriptase, DNA polymerase, DNA purification magnetic beads, nuclease-free water, anhydrous ethanol, and Qubit quantitative reagent.

8. A multiplex detection method for tropical pathogen spectrum based on tNGS technology, comprising: Extracting nucleic acid from the sample to be tested as a template; Performing multiplex PCR amplification using the primer set described in any one of claims 1 to 5; Use multiplex PCR amplification products to construct tNGS sequencing libraries; performing high-throughput sequencing on the sequencing library; The sequencing results are analyzed and compared with bioinformatics to determine the type of pathogen in the sample to be tested.

9. The method according to claim 8, characterized in that When the nucleic acid is RNA, reverse transcription is used to transcribe the RNA into cDNA.

10. The method according to claim 8, characterized in that The multiplex PCR amplification products are also subjected to one or both of purification and a second round of multiplex PCR amplification.

11. The method according to claim 8, characterized in that The high-throughput sequencing platform includes the MGI sequencing platform or the Illumina sequencing platform or other sequencing platforms with the same sequencing principle.

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