A multiplex detection primer set for tropical pathogens based on tNGS technology and its application

Abstract

The present invention discloses a multiplex detection primer set for tropical pathogens based on tNGS technology and its applications. The multiplex detection primer set includes primers shown in SEQ NO.: 1 to SEQ NO.: 260 in the sequence listing. Using this multiplex detection primer set, 100 common tropical pathogens can be efficiently and accurately detected in a single reaction system.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection, and in particular to a multiplex detection primer set for tropical pathogens based on tNGS technology and applications thereof. Background Art

[0002] The development of pathogen detection technology has evolved from traditional morphological and biochemical testing to modern molecular biology testing. With the rapid development of gene sequencing technology, high-throughput sequencing (NGS) has gradually become an important tool for pathogen detection. However, while traditional metagenomic sequencing (mNGS) can detect a wide range of pathogens, it struggles to meet the demands of rapid clinical testing due to high sequencing costs, complex data analysis, and the large sample size required. In recent years, the emergence of tNGS technology has provided a new solution for pathogen detection. tNGS significantly improves detection sensitivity and specificity by designing specific primers to capture and sequence target regions. However, existing tNGS technology still faces challenges in detecting multiple pathogens, including limited detection range, insufficient sensitivity, and complex testing procedures. Summary of the Invention

[0003] In response to the above-mentioned deficiencies in the prior art, the present invention proposes a multiplex detection primer set for tropical pathogens based on tNGS technology and its application.

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

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

[0006] In one embodiment, 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 herpes virus type 3, human herpes virus 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, new Bunyavirus, Cryptococcus neoformans, Coxsackievirus A16, Rhizopus, Acanthamoeba, Trichomonas, Mucorales, Hantavirus, Bartonella hansenii, Salmonella, Powassan virus, Burkholderia cepacia, Entamoeba histolytica, Bacillus anthracis, Taenia solium, monkey Poxvirus, 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 Staphylococci, 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 xenopus, Mycobacterium gordonii, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium ulcerans, Mycobacterium scrofula, Mycobacterium malmoe, Mycobacterium vaccae, Mycobacterium avium-intracellulare complex, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium haemophilus, Mycobacterium simian, Mycobacterium leprae.

[0007] In one embodiment, the four drug-resistant genes are blaCTX-M, mecA, mecC, and mecB.

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

[0009] The tropical pathogen spectrum multiplex detection kit provided by the present invention comprises the primer set.

[0010] In one embodiment, 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.

[0011] The present invention provides a multiplex detection method for tropical pathogens based on tNGS technology, comprising:

[0012] Extracting nucleic acid from the sample to be tested as a template;

[0013] Multiplex PCR amplification was performed using the primer sets described above;

[0014] Use multiplex PCR amplification products to construct tNGS sequencing libraries;

[0015] performing high-throughput sequencing on the sequencing library;

[0016] The sequencing results are analyzed and compared with biological information to determine the type of pathogen in the sample to be tested.

[0017] In one embodiment, when the nucleic acid is RNA, reverse transcription is used to transcribe the RNA into cDNA.

[0018] In one embodiment, the multiplex PCR amplification products are further subjected to one or both of purification and a second round of multiplex PCR amplification.

[0019] In one embodiment, the high-throughput sequencing platform includes a MGI sequencing platform or an Illumina sequencing platform or other sequencing platforms with the same sequencing principle.

[0020] The tropical pathogen spectrum multiplex detection primers based on tNGS technology proposed in this invention are designed by specific primers for gene fragments of common pathogenic microorganisms in tropical regions, targeting the target sequences of pathogens prevalent in tropical regions, and efficiently and accurately detecting 100 common pathogens in tropical regions in the same reaction system. DETAILED DESCRIPTION

[0021] Definition: tNGS (targeted Next-Generation Sequencing): Target region high-throughput sequencing is a technology that sequences specific gene regions and has the characteristics of high throughput, high sensitivity, and high specificity.

[0022] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to specific embodiments.

[0023] Example 1

[0024] Determine the range of common pathogens in tropical regions. Based on real epidemiological data from tropical regions, and by searching the literature and gaining consensus from experts in the field of infection, we determined the range of common pathogens in tropical regions. Ultimately, we identified 100 tropical pathogens, along with four drug-resistance genes and four Mycoplasma pneumoniae mutation sites for testing.

[0025] 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 spp., Angiostrongylus cantonensis, La Crosse virus, new Bunyavirus, Cryptococcus neoformans, Coxsackievirus A16, Rhizopus spp., Acanthamoeba spp., Trichomonas spp., Mucorales, Hantavirus, Bartonella henselae, Salmonella spp., Powassan virus, Burkholderia cepacia, Entamoeba histolytica, Bacillus anthracis, Taenia solium, Monkeypox virus, Malaria Protozoa, 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, Elizabeth II 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 toadense, 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 simian, Mycobacterium leprae.

[0026] The four drug-resistant genes are blaCTX-M, mecA, mecC, and mecB.

[0027] The four Mycoplasma pneumoniae mutation sites are 2063 / 2064 / 2067 / 2617 point mutations.

[0028] Specific primer design. Download high-quality pathogen genomes, construct a local genome database, use software to determine the specific genome sequence, and design primer pairs. Primer lengths should be set within the 18-25 bp range, with a GC content of 40%-60%, avoiding more than three consecutive identical bases.

[0029] Specific primer screening: Purchase standard strains, verify primer performance, and remove primers that cause primer dimers or low amplification efficiency.

[0030] The detection primer set for the target pathogenic hospital microorganisms was screened and obtained through the above development process, as shown in Table 1.

[0031] Table 1. Target pathogenic microorganisms and their corresponding tNGS primer set sequences provided by the present invention

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] Detecting patient samples using the above primer set includes the following steps:

[0039] Gently shake the sample to mix, pipette 800 μL of the corresponding sample, add 80 μL of lysis buffer L, and transfer to a pre-filled grinding tube. Place the grinding tube containing the sample into the cell wall disruptor and run the cell wall disruption program (6 M / S vibration intensity, 30 s grinding time, 30 s interval time, 6 cycles). After disruption, centrifuge at 12,000 rpm for 3 minutes to eliminate foam. Transfer 600 μL of the supernatant to well A of a deep-well plate. Finally, add 40 μL of 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 the automated extraction instrument. Start the pre-set program and start the automated extraction, which will run for approximately 30 minutes. After the program is completed, remove the deep-well plate, transfer the nucleic acid in well F to a 1.5 mL centrifuge tube, and measure the Qubit concentration.

[0040] Take a 0.2 mL PCR tube and prepare the reaction system according to Table 2 below.

[0041] Table 2 Multiplex PCR reaction system

[0042]

[0043] After the above system is prepared, vortex mix thoroughly, centrifuge briefly, place on a PCR instrument, and perform the reaction according to the program in Table 3 below.

[0044] Table 3 PCR amplification system

[0045]

[0046] Before use, equilibrate the DNA purification magnetic beads at room temperature for 30 minutes and mix thoroughly. Add 45µL of magnetic beads to the PCR product, vortex to mix thoroughly, incubate at room temperature for 5 minutes, centrifuge briefly, and place on a magnetic stand for 3 minutes. Discard the supernatant. Add 50µL of Clean Buffer, gently vortex to mix thoroughly, incubate at 37°C on a PCR machine for 5 minutes, centrifuge briefly, place on a magnetic stand for 3 minutes, and discard the supernatant. After discarding, use a 10µL pipette to remove the supernatant. Add 200µL of 80% ethanol, incubate for 30 seconds, and discard the supernatant. Repeat this process. Place the microcentrifuge tube on a magnetic stand, aspirate any excess liquid, and air dry at room temperature. Elute with 30µL of nuclease-free water, vortex to mix thoroughly, incubate at room temperature for 5 minutes, centrifuge briefly, and place on a magnetic stand for 3 minutes. Transfer 28µL of the supernatant to a new microcentrifuge tube (this is the tNGS library). Measure 1µL of the supernatant using a Qubit assay and record the concentration.

[0047] DNA nanospheres were prepared from 1 pmol of the mixed tNGS library using a one-step DNB preparation kit. The corresponding reagents were added to the sequencing reagent reservoir, and high-throughput sequencing was performed using the MGISEQ-20S gene sequencer with a read length of SE50. The raw data was first quality-controlled and adapters removed. The cleaned data was then analyzed using the bioinformatics analysis pipeline to obtain pathogen detection results.

[0048] To validate the detection performance of the primer set provided by the present invention, targeted pathogen high-throughput sequencing (tNGS) was performed on known pathogen-positive samples. A commercially available certified PCR product was used as a control, and the results were compared. The results are shown in Table 4 below.

[0049] Table 4. Comparison of results

[0050]

[0051] The results showed that tNGS had excellent detection performance, with no missed detections, which was consistent with the fluorescence PCR detection results.

[0052] Based on real-world epidemiological data from tropical regions, this method selects 100 tropical pathogens, covering regionally prevalent, high-threat pathogens (such as malaria, dengue fever, and Mycobacterium tuberculosis). This method addresses the problem of missed detections caused by traditional detection methods due to regional limitations. By combining analysis of four drug-resistance genes of greatest clinical concern with four mutation sites in Mycoplasma pneumoniae, patients can be provided with a precise, tailored medication plan, reducing the risk of overuse of broad-spectrum antibiotics.

[0053] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

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

2. A multiplex detection kit for tropical pathogens, comprising the primer set according to claim 1.

3. The kit according to claim 2, wherein 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.

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