CN115478114A - Specific primer group of mycobacteria and fungal gene and sequencing method of mycobacteria and fungal infection - Google Patents

Specific primer group of mycobacteria and fungal gene and sequencing method of mycobacteria and fungal infection Download PDF

Info

Publication number
CN115478114A
CN115478114A CN202210954187.3A CN202210954187A CN115478114A CN 115478114 A CN115478114 A CN 115478114A CN 202210954187 A CN202210954187 A CN 202210954187A CN 115478114 A CN115478114 A CN 115478114A
Authority
CN
China
Prior art keywords
nucleotide sequence
sequence shown
primer
seq
primers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210954187.3A
Other languages
Chinese (zh)
Inventor
逄宇
许佩松
王云飞
谷红仓
李姗姗
任卫聪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Shengting Medical Technology Co ltd
Beijing Chest Hospital
Original Assignee
Hangzhou Shengting Medical Technology Co ltd
Beijing Chest Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Shengting Medical Technology Co ltd, Beijing Chest Hospital filed Critical Hangzhou Shengting Medical Technology Co ltd
Publication of CN115478114A publication Critical patent/CN115478114A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
    • C12R2001/325—Mycobacterium avium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/67—Aspergillus flavus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/68—Aspergillus fumigatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/725—Candida albicans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/74—Candida tropicalis

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a mycobacterium and fungus gene specific primer set which comprises specific genes IS6100, hsp65, rpoB, gyrA, L1A1, RPB1, tub2, caM, URA5, CAP10, mtLSU, kex-1 or MP1 primer set. The invention utilizes the multiplex PCR technology to amplify the target genes of pathogenic bacteria to be detected in one reaction, and combines a nanopore sequencing platform to carry out high-throughput detection on specific gene amplicons, thereby simultaneously detecting 6 mycobacteria and 12 fungi.

Description

Specific primer group of mycobacteria and fungal gene and sequencing method of mycobacteria and fungal infection
Technical Field
The invention relates to the technical field of nanopore sequencing technology, in particular to a mycobacterium and fungal gene specificity primer group and a sequencing method of mycobacterium and fungal infection.
Background
Pulmonary disease patients infected by mycobacterium tuberculosis, nontuberculous mycobacteria and invasive fungi have higher similarity in clinical manifestations, manifested by cough, expectoration and fever. The imaging can also be manifested as lung shadow or lung cavity, which brings certain difficulties for clinical diagnosis and treatment. The traditional microorganism detection gold standard is pathogen culture, but the culture time of mycobacteria and fungi is long, the culture medium is harsh, the diagnosis is difficult to implement, and the treatment effect is possibly delayed. In recent years, molecular diagnostic techniques such as real-time fluorescent quantitative PCR and metagenomic sequencing (mNGS) have also begun to be widely used for clinical diagnosis. However, for patients with pulmonary diseases, real-time fluorescence quantitative PCR can diagnose Mycobacterium tuberculosis, but has no effective diagnostic measure for diagnosing non-Mycobacterium tuberculosis and fungi; although the metagenome sequencing technology can also comprehensively distinguish mycobacterium tuberculosis, nontuberculous mycobacterium and fungal infection, the detection cost is high, and the metagenome sequencing technology has certain limitation on the detection of low-abundance pathogenic bacteria. Currently, there is no economical, efficient, rapid, and accurate means to distinguish between mycobacterium tuberculosis, nontuberculous mycobacteria, and fungal infections in the clinic. At present, the effective differentiation of pathogenic bacteria caused by tuberculosis, non-tuberculosis and invasive fungi in pulmonary shadow is not carried out clinically, and the detection and differentiation of the bacteria relate to different treatment modes.
Nanopore sequencing technology is a new generation sequencing technology which is started in recent years, is a third generation sequencing technology (also called fourth generation sequencing technology) which integrates high throughput, high speed, ultra-long reading length and lower cost, and has great prospect in rapid diagnosis of pathogenic microorganisms based on the advantages. The current nanopore sequencing technology cannot detect mycobacterium tuberculosis, nontuberculous mycobacteria and invasive fungi.
Disclosure of Invention
The invention provides a mycobacterium and fungal gene specific primer group and a sequencing method using the primer group, aiming at overcoming the defect that the nanopore sequencing technology in the prior art can not detect mycobacterium tuberculosis, nontuberculous mycobacteria and invasive fungi, and having the advantages of simplicity, convenience, rapidness, comprehensiveness, accuracy and the like, and being beneficial to guiding the clinical differentiation of tuberculosis, nontuberculous and invasive fungal infection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a mycobacterium and fungal gene specific primer set comprises specific genes IS6100, hsp65, rpoB, gyrA, L1A1, RPB1, tub2, caM, URA5, CAP10, mtLSU, kex-1 or MP1 primer set, wherein the primer sequence of IS6100 comprises a nucleotide sequence shown in SEQ01 and comprises a nucleotide sequence shown in SEQ 02; or the primer set of hsp65 comprises a nucleotide sequence having the sequence shown in SEQ03 and comprises a nucleotide sequence having the sequence shown in SEQ 04; or the primer set of rpoB comprises a nucleotide sequence having the nucleotide sequence shown in SEQ05 and comprises a nucleotide sequence having the nucleotide sequence shown in SEQ 06; or the primer set of gyrA comprises a nucleotide sequence shown as SEQ07 and a nucleotide sequence shown as SEQ 08; or the primer set of L1A1 comprises a nucleotide sequence having a sequence shown as SEQ09 and comprises a nucleotide sequence having a sequence shown as SEQ 10; or the primer set of RPB1 comprises a nucleotide sequence shown in SEQ11 and a nucleotide sequence shown in SEQ 12; or the primer set of Tub2 comprises a nucleotide sequence having a sequence shown in SEQ13 and comprises a nucleotide sequence having a sequence shown in SEQ 14; or the primer set of CaM comprises a nucleotide sequence shown in SEQ15 and comprises a nucleotide sequence shown in SEQ 16; or the primer set of URA5 comprises a nucleotide sequence shown as SEQ17 and a nucleotide sequence shown as SEQ 18; or the primer set of CAP10 comprises a nucleotide sequence having the sequence shown in SEQ19 and comprises a nucleotide sequence having the sequence shown in SEQ 20; or the primer set of mtLSU comprises a nucleotide sequence having the nucleotide sequence shown in SEQ21 and comprises a nucleotide sequence having the nucleotide sequence shown in SEQ 20; or the primer set of Kex-1 comprises a nucleotide sequence shown in SEQ23 and comprises a nucleotide sequence shown in SEQ 24; or the MP1 primer group comprises a nucleotide sequence shown as SEQ25 and a nucleotide sequence shown as SEQ 26.
Preferably, the mycobacteria include mycobacterium tuberculosis and nontuberculous mycobacteria including: mycobacterium avium, M.intracellulare, M.cheloniae, M.abscessus, M.kansasii.
Preferably, the fungi include candida and aspergillus.
Preferably, the Candida species include Candida albicans, candida glabrata, candida tropicalis, and Candida parapsilosis.
Preferably, the aspergillus includes aspergillus fumigatus, aspergillus flavus, aspergillus niger, aspergillus terreus; the cryptococcus includes Cryptococcus gatherens, cryptococcus neoformans, malneffeta, and Pneumocystis jejuni.
The invention also discloses a sequencing method for detecting mycobacteria and fungal infection by using the specific primer group of the mycobacteria and fungal gene, which comprises the following steps:
1) Designing primers of IS6100, hsp65, rpoB and gyrA covering the mycobacterium specificity typing gene; covering candida specific typing gene L1A1 and RPB1 primers;
2) Adding a base filling sequence TTTCTGTTGGTGCTGATATTG to the 5 'end of each forward primer-F, and adding a base filling sequence ACTTGCCTGTCGCTCCTATCTTC to the 5' end of each reverse primer-R to obtain an amplification primer containing a second round PCR primer binding site;
3) Diluting the amplification primers of the second round PCR primer binding sites, and mixing forward and reverse primers of each gene; secondly, forming a primer pool by the mixed primers;
4) Synthesizing a Barcode label primer suitable for the second round of PCR, diluting all the Barcode dry powder primers, and mixing forward and reverse primers of the same Barcode to form a Barcode primer pool.
Preferably, the method further comprises a step 5), wherein the step 5) is used for extracting the genome DNA.
Preferably, the method further comprises a step 6), wherein the step 6) is to perform first round amplification and purification by using a primer pool.
Preferably, the method also comprises a step 7), wherein the step 7) is a second round of amplification and purification by using a Barcode primer.
Preferably, the method further comprises a step 8), wherein the step 8) is to construct a sequencing library and purify the sequencing library.
The beneficial effects of the invention are: (1) The invention has the advantages of simplicity, rapidness, comprehensiveness, accuracy and the like, and is favorable for guiding clinical differentiation of tuberculosis, non-tuberculosis and invasive fungal infection. (2) The invention utilizes the multiplex PCR technology to amplify the target gene of the pathogenic bacteria to be detected in one reaction, and combines a nanopore sequencing platform to carry out high-throughput detection on the specific gene amplicon, thereby simultaneously detecting 6 mycobacteria and 11 fungi.
Drawings
FIG. 1 is a schematic diagram of the library construction scheme of the present invention;
FIG. 2 is a diagram of agarose gel electrophoresis of 4 positive samples and 1 negative control sample after two rounds of amplification;
FIG. 3 is a qPCR identification profile of 4 different Mycobacterium tuberculosis copy number samples.
In the figure, NC represents water.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
Example 1: 1) Designed to cover 6 species of mycobacteria (including mycobacterium tuberculosis; non-tubercular Mycobacteria include: mycobacterium avium, mycobacterium intracellulare, mycobacterium cheloniae, mycobacterium abscessus, mycobacterium kansasii, but not limited to the above-mentioned nontuberculous mycobacteria) and 12 fungi (candida include: candida albicans, candida glabrata, candida tropicalis, candida parapsilosis, but are not limited; the aspergillus comprises: aspergillus fumigatus, aspergillus flavus, aspergillus niger, aspergillus terreus, but not limited thereto; cryptococcus include: cryptococcus gatherensis, cryptococcus neoformans; marneffei lanuginosa; yersinia) specific genes IS6100, hsp65, rpoB, gyrA, L1A1, RPB1, tub2, caM, URA5, CAP10, mtLSU, kex-1, MP1 primers, specific primer sequence information IS shown in table 1.
TABLE 1 primers specific for mycobacterial and fungal genes
Figure BDA0003790504870000031
Figure BDA0003790504870000041
2) Adding a base filling sequence TTTCTGTTGGTGCTGATATTG to the 5 'end of each forward primer-F, and adding a base filling sequence ACTTGCCTGTCGCTCCTATCTTC to the 5' end of each reverse primer-R to obtain an amplification primer containing a second round PCR primer binding site, wherein the specific primer sequence is shown in Table 2.
TABLE 2 amplification primers for drug-resistance-associated genes of Mycobacterium tuberculosis
Figure BDA0003790504870000042
Figure BDA0003790504870000051
3) Diluting all primers in the above Table 2 to 100. Mu.M, and mixing forward and reverse primers of each gene in equal proportion; secondly, forming a primer pool by the mixed primers, wherein the concentration ratio of the primers corresponding to IS6100, hsp65, rpoB, gyrA, L1A1, RPB1, tub2, caM, URA5, CAP10, mtLSU, kex-1 and MP1 in the primer pool IS 0.5:1:1.2:1:0.9:1.5:1: 1.2:1.5:1:0.8:1.2:1. Although primer mixtures of different ratios may also achieve the results of the invention, such ratios are preferred.
4) Barcode tag primers suitable for the second round of PCR are synthesized, each Barcode primer corresponds to one sample, and each sample is provided with different Barcode tags. All the barcode dry powder primers were diluted to 10 μ M and forward and reverse primers of the same barcode were mixed in equal proportion to form a barcode primer pool. Specific primer sequences are shown in Table 3.
TABLE 3 Bar code primer information Table
Primer name Primer sequence (5 '-3') Primer sequences
barcode01-F GGTGCTGAAGAAAGTTGTCGGTGTCTTTGTGTTAACCTTTCTGTTGGTGCTGATATTGC SEQ53
barcode01-R GGTGCTGAAGAAAGTTGTCGGTGTCTTTGTGTTAACCTACTTGCCTGTCGCTCTATCTT SEQ54
barcode02-F GGTGCTGTCGATTCCGTTTGTAGTCGTCTGTTTAACCTTTCTGTTGGTGCTGATATTGC SEQ55
barcode02-R GGTGCTGTCGATTCCGTTTGTAGTCGTCTGTTTAACCTACTTGCCTGTCGCTCTATCTT SEQ56
barcode03-F GGTGCTGGAGTCTTGTGTCCCAGTTACCAGGTTAACCTTTCTGTTGGTGCTGATATTGC SEQ57
barcode03-R GGTGCTGGAGTCTTGTGTCCCAGTTACCAGGTTAACCTACTTGCCTGTCGCTCTATCTT SEQ58
barcode04-F GGTGCTGTTCGGATTCTATCGTGTTTCCCTATTAACCTTTCTGTTGGTGCTGATATTGC SEQ59
barcode04-R GGTGCTGTTCGGATTCTATCGTGTTTCCCTATTAACCTACTTGCCTGTCGCTCTATCTT SEQ60
barcode05-F GGTGCTGCTTGTCCAGGGTTTGTGTAACCTTTTAACCTTTCTGTTGGTGCTGATATTGC SEQ61
barcode05-R GGTGCTGCTTGTCCAGGGTTTGTGTAACCTTTTAACCTACTTGCCTGTCGCTCTATCTT SEQ62
barcode06-F GGTGCTGTTCTCGCAAAGGCAGAAAGTAGTCTTAACCTTTCTGTTGGTGCTGATATTGC SEQ63
barcode06-R GGTGCTGTTCTCGCAAAGGCAGAAAGTAGTCTTAACCTACTTGCCTGTCGCTCTATCTT SEQ64
barcode07-F GGTGCTGGTGTTACCGTGGGAATGAATCCTTTTAACCTTTCTGTTGGTGCTGATATTGC SEQ65
barcode07-R GGTGCTGGTGTTACCGTGGGAATGAATCCTTTTAACCTACTTGCCTGTCGCTCTATCTT SEQ66
barcode08-F GGTGCTGTTCAGGGAACAAACCAAGTTACGTTTAACCTTTCTGTTGGTGCTGATATTGC SEQ67
barcode08-R GGTGCTGTTCAGGGAACAAACCAAGTTACGTTTAACCTACTTGCCTGTCGCTCTATCTT SEQ68
barcode09-F GGTGCTGAACTAGGCACAGCGAGTCTTGGTTTTAACCTTTCTGTTGGTGCTGATATTGC SEQ69
barcode09-R GGTGCTGAACTAGGCACAGCGAGTCTTGGTTTTAACCTACTTGCCTGTCGCTCTATCTT SEQ70
barcode10-F GGTGCTGAAGCGTTGAAACCTTTGTCCTCTCTTAACCTTTCTGTTGGTGCTGATATTGC SEQ71
barcode10-R GGTGCTGAAGCGTTGAAACCTTTGTCCTCTCTTAACCTACTTGCCTGTCGCTCTATCTT SEQ72
barcode11-F GGTGCTGGTTTCATCTATCGGAGGGAATGGATTAACCTTTCTGTTGGTGCTGATATTGC SEQ73
barcode11-R GGTGCTGGTTTCATCTATCGGAGGGAATGGATTAACCTACTTGCCTGTCGCTCTATCTT SEQ74
barcode12-F GGTGCTGCAGGTAGAAAGAAGCAGAATCGGATTAACCTTTCTGTTGGTGCTGATATTGC SEQ75
barcode12-R GGTGCTGCAGGTAGAAAGAAGCAGAATCGGATTAACCTACTTGCCTGTCGCTCTATCTT SEQ76
Example 2
The method comprises the following steps: the first round of multiplex PCR enriched the specific genes of 6 mycobacteria and 12 fungi in the sample.
1) 4 DNA samples (M.tuberculosis, M.avium, aspergillus fumigatus mixed nucleic acid) with different copy numbers of M.tuberculosis (FIG. 1) were subjected to PCR amplification (plus 1 PCR negative control) using the mixed primer pool: the total reaction volume was 50. Mu.l, 2 XPUSIONHFBufferPCRMastermix (NEB) 25. Mu.l, 5. Mu.l of optimized primer pool, 50ng of genomic DNA, and 50. Mu.l of ddH2O supplemented. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 15s, annealing at 65 ℃ for 60s, reduction by 1 ℃ per cycle, extension at 72 ℃ for 15s,10 cycles; denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 60s, extension at 72 ℃ for 15s,20 cycles; after the circulation is finished, the product is extended for 5min at 72 ℃ and stored at 4 ℃.
2) Adding 30 mu l of AMpureXPbeads into 50 mu l of the multiple PCR product, uniformly blowing and stirring, and standing at room temperature for 5min to fully combine the magnetic beads and the PCR product;
placing the purification system on a magnetic frame, waiting for 2-5min until magnetic beads are completely adsorbed on one side of the magnetic frame, and removing supernatant by using a pipettor;
adding 200 mul of freshly prepared 80% ethanol, standing for 30s on a magnetic frame, removing the supernatant, and washing once again to fully remove impurities;
fully removing residual ethanol by using a small-range pipettor, uncovering the cover, and air-drying at room temperature for 5min until the magnetic beads are dried;
taking down the purification tube from the magnetic frame, adding 25 mu lddH2O to elute DNA, blowing and uniformly mixing to ensure that the magnetic beads are in a complete suspension state, standing for 3min at room temperature, and fully eluting DNA;
and placing the purification tube on the magnetic frame again, standing for 3min, and sucking 23 mu l of elution liquid for the second round of amplification when the magnetic beads are completely adsorbed on one side of the magnetic frame.
Step two: two-round PCR amplification
1) Two rounds of PCR amplification were performed with Barcode tag primers (BC 01-BC 05) to bring different samples with different tags for sequencing on a mix-top machine. The reaction system was 50. Mu.l, 2 XPUSIONHFBufferPCRMastermix (NEB) 25. Mu.l, barcode primer 2. Mu.l, and first amplification purification product 23. Mu.l. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 15s, annealing at 62 ℃ for 15s, extension at 72 ℃ for 30s,6 cycles; after circulation, the product is extended at 72 deg.C for 5min and stored at 4 deg.C.
2) The magnetic bead purification method was identical to the purification method in step one, and at the end of the elution of DNA, 27. Mu.l of DNA was eluted with 30. Mu.lddH 2O and used for library construction.
3) The concentration of the purified product is measured by the concentration of Qubit4.0, which is respectively as follows: 76.5 ng/. Mu.l sample No. 1, 73.2 ng/. Mu.l sample No. 2, 52.9 ng/. Mu.l sample No. 3, 26.1 ng/. Mu.l sample No. 4, 0.38 ng/. Mu.l sample No. 5.
Step three: library construction
1) Library mixing (pooling): the 5 purified products after two rounds of amplification were pooling (10. Mu.l for less than 100 ng) per sample at 100ng, and then the total volume was supplemented to 50. Mu.l with ddH 2O.
2) End repair and 3' end add a tail: the total reaction system was 65. Mu.l, the mixed DNA solution was 50. Mu.l, and the end-modifying mix (Vazyme) was 15. Mu.l. The reaction procedure is as follows: reaction at 30 ℃ for 15min, reaction at 65 ℃ for 15min, and storage at 4 ℃.
3) And (3) purifying a terminal repair product: after adding 39. Mu.l of AMpureXPBeads to 65. Mu.l of PCR product, the DNA was purified in the same manner as the magnetic bead purification in step one, and finally eluted with 30. Mu.lddH 2O, and 25. Mu.l of the DNA was used for the linker ligation reaction.
4) And (3) connecting a nanopore sequencing joint: the total reaction system was 50. Mu.l, 25. Mu.l of the purified product of end-repair, 10. Mu.l of 5 XLigationBuffer (NEB), 8. Mu.l of T4RapidDNAligase (NEB), 1. Mu.l of Adaptermix (Nanopore), and 6. Mu.l of ddH 2O. The reaction procedure is as follows: reaction at 20 ℃ for 15min and storage at 4 ℃.
5) Purifying a joint connection product: adding 30 mul of AMpureXPbeads into 50 mul of the joint connecting product, uniformly blowing and stirring, and standing at room temperature for 5min to ensure that the magnetic beads are fully combined with the PCR product; placing the purification system on a magnetic frame, waiting for 2-5min until the magnetic beads are completely adsorbed on one side of the magnetic frame, and removing the supernatant by using a liquid transfer device; adding 200 μ l SFB (supplied by NanoporeLSK109 kit, LFB is not used), standing on a magnetic frame for 30s, discarding the supernatant, and repeating once; sucking to remove residual SFB, uncovering the cover, drying at room temperature for about 5min, and adding 15 mu lddH2O to elute DNA after the magnetic beads are dried; and (4) uniformly mixing the materials by blowing, standing for 3min at room temperature, and sucking 13 mu l to obtain a final library.
6) The final library concentration was determined using Qubit: 15.6 ng/. Mu.l.
Step four: nanopore sequencing Using a MinION sequencer (sequencing kit: SQK-LSK109, sequencing chip: R9) from Nanopore corporation
1) The R9 chip is subjected to chip activity induction at least 5min before a sample is added into the chip, so that the preservation solution in the chip is completely replaced by the sequencing buffer solution;
2) The library loading method is carried out according to official instructions of Oxford nanopore;
3) Setting a sequencer: the sequencing kit selects SQK-LSK109, selects trimbarcode, and selects a Highaccuracy mode in a sequencing mode to perform nanopore sequencing.
Example 3
The database analysis and comparison process comprises the following steps:
and after the sequencing data is off the machine, automatically converting the sequencing result by using the nanopore sequencer to generate a sequencing file in a fastq format for subsequent analysis.
1) And (3) data quality control: performing quality control analysis on the offline data, and selecting Qscore more than or equal to 9 to remove low-quality reads and connector sequences in the quality control process;
2) Filtering the sequence with the sequence length less than 450bp or more than 750bp to obtain a target sequence which accords with the expected design;
3) Comparing the target sequence with mycobacterial and fungal genome sequences in a database;
4) The species detected and the number of sequences were generated as shown in Table 4.
TABLE 4.5 sample distribution
Figure BDA0003790504870000081
The present invention performed specific gene detection of mycobacteria and fungi ( sample numbers 1,2,3, 4) on 4 positive samples with different copy numbers (fig. 2, fig. 3), and the lower the copy number of the sample, the higher the product concentration of the two-round PCR, the higher the number of detected sequences and the number of uniquely aligned sequences.
In order to verify the accuracy of the invention on the detection results of mycobacterium tuberculosis, nontuberculous mycobacteria and fungus positive samples. This example was performed using the present invention on 18 different positive samples. These samples were verified by one-generation sequencing and real-time fluorescent quantitative PCR, as shown in Table 5.
Table 5.18 sample accuracy verification results
Figure BDA0003790504870000082
Figure BDA0003790504870000091
To verify the specificity of the present invention for the detection of M.tuberculosis, M.nontuberculosis and fungi. In this example, 17 specimens of negative bacteria such as Mycobacterium tuberculosis and nontuberculous mycobacteria, which are not pathogenic bacteria, were tested by the present invention. These samples were verified by one-generation sequencing verification, as shown in table 6. The results of specificity verification of 17 samples found that the results of cross-validation were ideal and no false positive results were found
Table 6.17 sample specificity verification results
Sample numbering Verification method and results Test results of the invention Number of sequences
1 Sanger streptococcus pneumoniae Negative of 0
2 Sanger staphylococcus aureus Negative of 0
3 Sanger haemophilus influenzae Negative of 0
4 Sanger Nocardia Negative of 0
5 Sanger E.coli Negative of 0
6 Sanger staphylococcus epidermidis Negative of 0
7 Sanger Pseudomonas aeruginosa Negative of 0
8 Sanger pseudomonas aeruginosa Negative of 0
9 Sanger Acinetobacter baumannii Negative of 0
10 Sanger maltophilia Negative of 0
11 Sanger Chlamydia psittaci Negative of 0
12 Sanger suppurative natureStreptococcus sp Negative of 0
13 Sanger Legionella pneumophila Negative of 0
14 qPCR human parainfluenza virus Negative of 0
15 qPCR influenza A virus Negative of 0
16 qPCR influenza virus Negative of 0
17 qPCR adenovirus Negative of 0
The above results indicate that the present invention can also ensure the efficacy of each primer by mixing these primers together.
The above description is only for the preferred embodiment of the present invention and is not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the present specification and the drawings, which are directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (10)

1. A mycobacterium and fungal gene specific primer set IS characterized by comprising a specific gene IS6100, hsp65, rpoB, gyrA, L1A1, RPB1, tub2, caM, URA5, CAP10, mtLSU, kex-1 or MP1 primer set, wherein the primer sequence of IS6100 comprises a nucleotide sequence shown in SEQ01 and comprises a nucleotide sequence shown in SEQ 02; or the primer set of hsp65 comprises a nucleotide sequence having the sequence shown in SEQ03 and comprises a nucleotide sequence having the sequence shown in SEQ 04; or the primer set of rpoB comprises a nucleotide sequence having a sequence shown in SEQ05 and comprises a nucleotide sequence having a sequence shown in SEQ 06; or the primer set of gyrA comprises a nucleotide sequence shown as SEQ07 and a nucleotide sequence shown as SEQ 08; or the primer set of L1A1 comprises a nucleotide sequence having the sequence shown in SEQ09 and comprises a nucleotide sequence having the sequence shown in SEQ 10; or the primer set of RPB1 comprises a nucleotide sequence having a sequence shown in SEQ11 and comprises a nucleotide sequence having a sequence shown in SEQ 12; or the primer set of Tub2 comprises a nucleotide sequence shown as SEQ13 and comprises a nucleotide sequence shown as SEQ 14; or the primer group of CaM comprises a nucleotide sequence shown as SEQ15 and a nucleotide sequence shown as SEQ 16; or the primer set of URA5 comprises a nucleotide sequence having the sequence shown in SEQ17 and comprises a nucleotide sequence having the sequence shown in SEQ 18; or the primer set of CAP10 comprises a nucleotide sequence having the sequence shown in SEQ19 and comprises a nucleotide sequence having the sequence shown in SEQ 20; or the primer set of mtLSU comprises a nucleotide sequence having a sequence shown in SEQ21 and comprises a nucleotide sequence having a sequence shown in SEQ 20; or the primer set of Kex-1 comprises a nucleotide sequence shown as SEQ23 and comprises a nucleotide sequence shown as SEQ 24; or the MP1 primer group comprises a nucleotide sequence shown as SEQ25 and a nucleotide sequence shown as SEQ 26.
2. The primer set of claim 1, wherein said mycobacteria comprise mycobacterium tuberculosis and nontuberculous mycobacteria, said nontuberculous mycobacteria comprise: mycobacterium avium, mycobacterium intracellulare, mycobacterium cheloniae, mycobacterium abscessus, mycobacterium kansasii.
3. The primer set of claim 1, wherein the fungus includes Candida and Aspergillus.
4. The set of mycobacterial and fungal gene-specific primers according to claim 3, wherein said Candida species comprises Candida albicans, candida glabrata, candida tropicalis, and Candida parapsilosis.
5. The primer set specific for mycobacterial and fungal genes of claim 4, wherein said Aspergillus comprises Aspergillus fumigatus, aspergillus flavus, aspergillus niger, aspergillus terreus; the cryptococcus includes Cryptococcus gatherens, cryptococcus neoformans, malneffeta, and Pneumocystis jecorii.
6. A method for sequencing mycobacterial and fungal infections using a set of mycobacterial and fungal gene-specific primers according to any one of claims 1 to 5; the method comprises the following steps:
1) Designing primers of IS6100, hsp65, rpoB and gyrA covering the mycobacterium specificity typing gene; covering candida specific typing gene L1A1 and RPB1 primers;
2) Adding a base filling sequence TTTCTGTTGGTGCTGATATTG to the 5 'end of each forward primer-F, and adding a base filling sequence ACTTGCCTGTGTCTCCTATCTTC to the 5' end of each reverse primer-R to obtain an amplification primer containing a second round PCR primer binding site;
3) Diluting the amplification primers of the second round PCR primer binding sites, and mixing forward and reverse primers of each gene; secondly, forming a primer pool by the mixed primers;
4) Synthesizing a Barcode label primer suitable for the second round of PCR, diluting all the Barcode dry powder primers, and mixing forward and reverse primers of the same Barcode to form a Barcode primer pool.
7. The sequencing method for detecting mycobacterial and fungal infections using the set of mycobacterial and fungal gene-specific primers of claim 6, further comprising step 5), wherein said step 5) is extracting genomic DNA.
8. The sequencing method for detecting mycobacterial and fungal infections using the set of mycobacterial and fungal gene-specific primers of claim 6, further comprising step 6), wherein step 6) is a first round of amplification and purification using a pool of primers.
9. The sequencing method for detecting mycobacterial and fungal infections using the set of mycobacterial and fungal gene-specific primers of claim 6, further comprising step 7), wherein step 7) is a second round of amplification and purification using Barcode primers.
10. The sequencing method for detecting mycobacterial and fungal infections using the set of mycobacterial and fungal gene-specific primers of claim 6, further comprising step 8), wherein step 8) is to construct a sequencing library and purify.
CN202210954187.3A 2022-07-29 2022-08-10 Specific primer group of mycobacteria and fungal gene and sequencing method of mycobacteria and fungal infection Pending CN115478114A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210909396 2022-07-29
CN2022109093966 2022-07-29

Publications (1)

Publication Number Publication Date
CN115478114A true CN115478114A (en) 2022-12-16

Family

ID=84423160

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210954187.3A Pending CN115478114A (en) 2022-07-29 2022-08-10 Specific primer group of mycobacteria and fungal gene and sequencing method of mycobacteria and fungal infection

Country Status (1)

Country Link
CN (1) CN115478114A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117070668A (en) * 2023-10-17 2023-11-17 江苏美克医学技术有限公司 Aspergillus flavus detection primer set, kit and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117070668A (en) * 2023-10-17 2023-11-17 江苏美克医学技术有限公司 Aspergillus flavus detection primer set, kit and application thereof
CN117070668B (en) * 2023-10-17 2023-12-26 江苏美克医学技术有限公司 Aspergillus flavus detection primer set, kit and application thereof

Similar Documents

Publication Publication Date Title
JP5596891B2 (en) Probe set, probe fixing carrier, and genetic testing method
JP5196845B2 (en) Probe set, probe carrier and inspection method
EP1803823B1 (en) Probe set, probe immobilized carrier and gene examination method
JP5196853B2 (en) Probe set, probe carrier and inspection method
CN112501268B (en) Nanopore sequencing-based primer group and kit for rapidly identifying respiratory microorganisms and application of primer group and kit
JP2008118912A (en) Probe, probe set, probe-immobilized carrier and method for testing gene
JP2008118914A (en) Probe, probe set, probe-immobilized carrier and genetic testing method
JP2008118905A (en) Probe, probe set, probe-immobilized carrier and methd for testing gene
JP2004313181A (en) Probe for detecting infection-inducing microbe, probe set, carrier and method for testing gene
JP2008118908A (en) Probe, probe set, probe-immobilized carrier and method for testing gene
WO2017193833A1 (en) Method and kit comprising 4,000 human pathogenic target genes
CN106191311B (en) A kind of multiple liquid phase genetic chip method and reagent of quick detection cavy LCMV, SV, PVM, Reo-3 virus
CN115478114A (en) Specific primer group of mycobacteria and fungal gene and sequencing method of mycobacteria and fungal infection
CN107354207B (en) liquid phase hybridization capture kit based on double-stranded probe, washing kit and application thereof
CN116287357A (en) Respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing
CN109337956B (en) Design method and kit for enriching multi-gene stem-loop probe based on NGS technology
CN113667729A (en) Rapid microorganism identification method based on nanopore sequencer
CN113265452A (en) Bioinformatics pathogen detection method based on Nanopore metagenome RNA-seq
CN112195238A (en) Primer group and kit for amplifying PKD1 gene
CN115873992A (en) Primer combination for detecting blood stream infection pathogen and application thereof
CN107674920B (en) Chimeric multiplex PCR primer composition and detection method
CN110791573B (en) Microsatellite locus and primer suitable for identifying golden monkey individual
JPH03206899A (en) Nucleic acid probe and method for use in detection of cryptocox neoholmance
CN109750098B (en) ATP7B gene large fragment deletion detection kit and detection method
CN113667728B (en) Quick mycobacterium identification method based on nanopore sequencer

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination