CN114480691A - Method and kit for detecting mycobacterium tuberculosis complex flora based on melting curve - Google Patents
Method and kit for detecting mycobacterium tuberculosis complex flora based on melting curve Download PDFInfo
- Publication number
- CN114480691A CN114480691A CN202210198175.2A CN202210198175A CN114480691A CN 114480691 A CN114480691 A CN 114480691A CN 202210198175 A CN202210198175 A CN 202210198175A CN 114480691 A CN114480691 A CN 114480691A
- Authority
- CN
- China
- Prior art keywords
- gyra
- rpob
- seq
- mpb64
- gene
- 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
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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- 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
- 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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention relates to the field of biological detection, in particular to a method and a kit for detecting mycobacterium tuberculosis complex flora based on a melting curve. The invention provides a multiplex primer, which comprises a specific primer pair and a specific probe of a mycobacterium tuberculosis insertion sequence IS6110, an MPB64 gene, an rpoB gene, a katG gene, an inhA gene and a gyrA gene. The invention also provides a corresponding kit and a detection method. The detection method can quickly identify the mycobacterium tuberculosis complex, can simultaneously judge the drug resistance of the mycobacterium tuberculosis complex to isoniazid, rifampicin and fluoroquinolone drugs, and has good application prospect.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a method and a kit for detecting mycobacterium tuberculosis complex flora based on a melting curve.
Background
Tuberculosis is a chronic infectious disease caused by infection with mycobacterium tuberculosis. Tuberculosis becomes a global serious public health problem, and multi-drug resistant tuberculosis and wide drug resistant tuberculosis are one of the problems to be solved urgently in clinic at present. Therefore, the rapid and accurate detection of Mycobacterium tuberculosis in a specimen is crucial to the control of tuberculosis.
Rifampin is a rifamycin semi-synthetic broad-spectrum antibacterial drug, which has obvious bactericidal effect on Mycobacterium Tuberculosis (MTB) and part of nontuberculous mycobacteria (including mycobacterium leprae, etc.) inside and outside host cells. The drug has been a key drug in a tuberculosis chemotherapy scheme since the invention in 1971, and rifampicin resistance is a multi-drug resistant tuberculosis marker, so that the detection of rifampicin resistance of mycobacterium tuberculosis is very necessary.
Fluoroquinolones are also called pyridonecids, and belong to artificial chemical synthesis antibacterial drugs. The clinically common fluoroquinolones mainly include norfloxacin, Pefloxacin (Pefloxacin norfloxacin), Enoxacin (Enoxacin fluazinam), Ofloxacin (Ofloxacin) and Ciprofloxacin (Ciprofloxacin). In recent years, new polyfluorinated quinolone varieties such as Lomefloxacin (Lomefloxacin), Fleroxacin (Fleroxacin polyfluoropericic acid), and Difloxacin (Difloxacin difluopericic acid) have been developed and marketed. With the increase of the use of quinolone antibacterial drugs in mycobacterium tuberculosis infection, the drug resistance rate of mycobacterium tuberculosis to the antibacterial drugs gradually rises. Therefore, detection of fluoroquinolone drug-resistant mutations has become necessary.
Isoniazid, also known as 4-pyridine formhydrazide and isoniazid, has high selectivity and strong inhibition and killing effects on mycobacterium tuberculosis, has good biological membrane penetrability, and is a preferred antitubercular drug due to good curative effect, low toxicity, low price and convenient oral administration. Since isoniazid has been used for about fifty years and some patients have developed resistance to infected tubercle bacillus, it is necessary to detect mutations in isoniazid resistance.
Currently, the clinically used drug resistance detection methods can be classified into phenotypic detection and molecular detection. The phenotype detection method is based on culture, detects the drug resistance of the mycobacterium tuberculosis by comparing and observing the growth condition of the mycobacterium tuberculosis in a culture medium containing or not containing drugs, and is the 'gold standard' of the current drug resistance detection. Most commonly used phenotypic tests require pure cultures of M.tuberculosis, the slow growth characteristics of which determine the long time required for the procedure and may lead to uncertainty in the results due to poor growth or other microbial contamination.
The existing genotype detection methods comprise a real-time fluorescence quantitative PCR method, a gene chip method, a reverse hybridization method, a DNA sequencing method and the like, but have the defects of complicated operation steps, higher requirements on the technical level of personnel, expensive equipment, incapability of detecting all mutation regions and the like. Therefore, there is a need in the art to develop a method for simultaneously detecting multi-drug resistant mycobacterium tuberculosis, which is simple and rapid to operate, has high sensitivity and strong specificity.
Disclosure of Invention
The invention aims to provide a method and a kit for detecting mycobacterium tuberculosis complex groups based on a melting curve.
To this end, in a first aspect, the present invention provides a multiplex primer for detecting Mycobacterium tuberculosis complex flora, comprising a specific primer pair having insertion sequences IS6110, MPB64 gene, rpoB gene, katG gene, inhA gene and gyrA gene; the sequence of the specific primer pair is as follows:
F-IS6110：CTCAGCGGATTCTTCGGTCGTGGTC(SEQ ID NO：1)；
R-IS6110：TGAGGTCGCCCGTCTACTTGGTGTT(SEQ ID NO：2)；
F-MPB64：ACATCAGCCTGCCCAGTTACTACCC(SEQ ID NO：3)；
R-MPB64：TCAGCCTGCCACAGCGTGTCATA(SEQ ID NO：4)；
F-rpoB：TGGAGGCGATCACACCGCAGAC(SEQ ID NO：5)；
R-rpoB：GTGCACGTCGCGGACCTCCAGCC(SEQ ID NO：6)；
F-katG：TGGAGCAGATGGGCTTGGG(SEQ ID NO：7)；
R-katG：CCAGCAGGGCTCTTCGTCA(SEQ ID NO：8)；
F-inhA：CGGAAATCGCAGCCACGTTAC(SEQ ID NO：9)；
R-inhA：ACGGGATACGAATGGGGGTTTG(SEQ ID NO：10)；
F-gyrA：GCAATGTTCGATTCCGGCTTCC(SEQ ID NO：11)；
R-gyrA：CGGGCTTCGGTGTACCTCAT(SEQ ID NO：12)。
further, the multiplex primers further comprise specific probes for insertion sequences IS6110, MPB64 gene, rpoB gene, katG gene, inhA gene and gyrA gene.
Further, the sequence of the specific probe is as follows:
P-IS6110：CGGCGCACCCACTTACGCCG(SEQ ID NO：13)；
P-MPB64：CTGGAAAATTACATCGCCCAG(SEQ ID NO：14)；
P1-rpoB：CATTGGCACCAGCCAGCTGAGCCAATG(SEQ ID NO：15)；
P2-rpoB：CCTGCTTCATGGACCAGAACAACCCGCTGGCAGG(SEQ ID NO：16)；
P3-rpoB：CCTGCTCGGGGTTGACCCACAAGCGCGCAGG(SEQ ID NO： 17)；
P4-rpoB：CCATGGCGACTGTCGGCGCTGCCATGG(SEQ ID NO：18)；
P-katG：CCTGCGGACGCGATCACCAGCGGCAGCAGG(SEQ ID NO：19)；
P-inhA：CCTGCCCCGACAACCTATCGTCTCGCCGCAGG(SEQ ID NO： 20)；
P1-gyrA：CGACCGCAGCCACGCCAAGTCGGTC(SEQ ID NO：21)；
P2-gyrA：CCCCGTTCAGTTGCCGAGACCATGGGG(SEQ ID NO：22)；
P3-gyrA：CCTGCCAACTACCACCCGCACGGCGACGCGGCAGG(SEQ ID NO：23)；
P4-gyrA：CCTGCTCGATCTACGACAGCCTGGTGCGGCAGG(SEQ ID NO：24)；
P5-gyrA：CATGGCCCTGCCCTGGTCGCTGCGCCATG(SEQ ID NO：25)。
further, the 5' end of the specific probe is modified with a fluorescent group selected from FAM, HEX, ROX, CY5, Cy3, TET, JOE and VIC; the 3' end of the specific probe is modified with a quenching group selected from BHQ, Dabcy1, TAMRA and MGB.
In some embodiments, the probe specific for insertion sequence IS6110 IS modified at the 5 'end with CY5 fluorophore and at the 3' end with BHQ2 quencher;
the specific probe of the MPB64 gene is modified with CY5 fluorescent group at the 5 'end and modified with BHQ2 quenching group at the 3' end;
the specific probe of the rpoB gene is modified with FAM fluorescent group at the 5 'end and modified with BHQ1 quenching group at the 3' end;
the specific probe of the katG gene is modified with a ROX fluorescent group at the 5 'end and modified with a BHQ2 quenching group at the 3' end;
the specific probe of the inhA gene is modified with ROX fluorescent group at the 5 'end and BHQ2 quenching group at the 3' end;
the specific probe of the gyrA gene is modified with ROX or VIC fluorescent group at the 5 'end and BHQ1 or BHQ2 quenching group at the 3' end.
Further, the specific probe comprises one or both of the following modifications: the modification is Peptide Nucleic Acid (PNA) and Locked Nucleic Acid (LNA).
In some embodiments, the underlined base modifications in the following specific probes are Locked Nucleic Acids (LNAs):
P1-rpoB：CATTGGCACCAGCCAGCTGAGCCAATG；
P2-rpoB：CCTGCTTCATGGACCAGAACAACCCGCTGGCAGG；
P-inhA：CCTGCCCCGACAACCTATCGTCTCGCCGCAGG；
P2-gyrA：CCCCGTTCAGTTGCCGAGACCATGGGG。
the second aspect of the invention provides the application of the multiplex primer in the detection of mycobacterium tuberculosis complex, which comprises the steps of taking the DNA of a sample to be detected as a template, and carrying out amplification and melting curve detection by using the multiplex primer.
In a third aspect of the invention, the application of the multiple primers in preparing a reagent for detecting the mycobacterium tuberculosis complex is provided.
The fourth aspect of the invention provides a kit for detecting mycobacterium tuberculosis complex flora, which comprises dNTPs, DNA polymerase and (NH)4)2SO4、MgCl2KCl, Tris-HCl and the multiple primer of the invention.
In some embodiments, the kit comprises a system a comprising: dNTPs, (NH)4)2SO4、MgCl2、KCl、Tris-HCl、F-IS6110、R-IS6110、 F-MPB64、R-MPB64、F-rpoB、R-rpoB、F-katG、R-katG、F-inhA、R-inhA、 F-gyrA、R-gyrA、P-IS6110、P-MPB64、P1-rpoB、P3-rpoB、P-katG、P-inhA、 P2-gyrA、P4-gyrA；
The system B comprises: dNTPs, (NH)4)2SO4、MgCl2、KCl、F-IS6110、R-IS6110、F-MPB64、R-MPB64、F-rpoB、R-rpoB、F-gyrA、R-gyrA、P-IS6110、P-MPB64、 P2-rpoB、P4-rpoB、P1-gyrA、P3-gyrA、P5-gyrA。
Further, the DNA polymerase is a hot start DNA polymerase.
Further, the working concentration of the DNA polymerase is 0.05-0.2U/muL; for example, 0.05U/. mu.L, 0.1U/. mu.L, 0.15U/. mu.L, 0.2U/. mu.L, etc.
Further, the concentration of the dNTPs is 0.15-0.25 mM; for example, 0.15mM, 0.2mM, 0.25mM, etc.
Further, the concentration of each specific primer is independently selected from 0.02-0.5 μmol/L; for example, 0.02. mu. mol/L, 0.05. mu. mol/L, 0.1. mu. mol/L, 0.15. mu. mol/L, 0.2. mu. mol/L, 0.25. mu. mol/L, 0.3. mu. mol/L, 0.35. mu. mol/L, 0.4. mu. mol/L, 0.45. mu. mol/L, 0.5. mu. mol/L, etc.
Further, the concentration of each specific probe is independently selected from 0.02-0.5 μmol/L; for example, 0.02. mu. mol/L, 0.05. mu. mol/L, 0.1. mu. mol/L, 0.15. mu. mol/L, 0.2. mu. mol/L, 0.25. mu. mol/L, 0.3. mu. mol/L, 0.35. mu. mol/L, 0.4. mu. mol/L, 0.45. mu. mol/L, 0.5. mu. mol/L, etc.
Further, said (NH)4)2SO4In a concentration of 5-15 mM; for example, 5mM, 10mM, 15mM, etc.
Further, the MgCl2Is 2-5 mM; for example, 2mM, 2.5mM, 3mM, 4mM, 5mM, etc.
Further, the concentration of the KCl is 5-15 mM; for example, 5mM, 10mM, 15mM, etc.
Further, the concentration of the Tris-HCl is 10-30 mM; e.g., 10mM, 20mM, 30mM, etc.; the pH value of the Tris-HCl is 8-8.5; such as 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, etc.
Further, the kit also comprises a negative quality control product and a positive quality control product; for example: the negative quality control material is water or normal saline; the positive quality control product contains the nucleotide sequences of the insertion sequences IS6110 and MPB64 genes and cannot be amplified by the specific primer pairs of the rpoB gene, the katG gene, the inhA gene and the gyrA gene.
In some embodiments, the kit further comprises a DNA extraction solution for extracting DNA from a sample to be tested.
Further, the DNA extracting solution comprises disodium ethylene diamine tetraacetate, Tris and TritonX-100.
In a fifth aspect of the present invention, a method for detecting mycobacterium tuberculosis complex flora is provided, which comprises the following steps:
(1) extracting DNA from a sample to be detected;
(2) and (2) performing PCR amplification and melting curve detection by using the DNA extracted in the step (1) as a template and using the multiplex primer.
The detection method is used for non-disease diagnostic or therapeutic purposes.
Further, the PCR amplification comprises the following two reaction systems:
dNTPs、(NH4)2SO4、MgCl2、KCl、Tris-HCl、F-IS6110、R-IS6110、F-MPB64、 R-MPB64、F-rpoB、R-rpoB、F-katG、R-katG、F-inhA, R-inhA, F-gyrA, R-gyrA, P-IS6110, P-MPB64, P1-rpoB, P3-rpoB, P-katG, P-inhA, P2-gyrA, P4-gyrA, DNA polymerase and template;
dNTPs、(NH4)2SO4、MgCl2KCl, F-IS6110, R-IS6110, F-MPB64, R-MPB64, F-rpoB, R-rpoB, F-gyrA, R-gyrA, P-IS6110, P-MPB64, P2-rpoB, P4-rpoB, P1-gyrA, P3-gyrA, P5-gyrA, DNA complex enzyme and template.
Further, the reaction conditions of the PCR amplification and the melting curve detection comprise:
S1：95℃10min；
s2: 25s at 95 ℃, 25s at 70 ℃ (1 ℃ reduction per cycle), 30s at 72 ℃, 13 cycles;
s3: collecting fluorescence signals at 95 ℃ for 25s, 58 ℃ for 25s, 72 ℃ for 30s, 42 cycles, and 72 ℃ for 30 s;
S4：95℃1min，45℃1min；
s5: fluorescence signals were collected at 45-85 ℃ every 1 ℃.
According to the detection method provided by the invention, whether the sample to be detected contains the mycobacterium tuberculosis complex flora and whether the mycobacterium tuberculosis complex flora has the tolerance of rifampicin, isoniazid and/or fluoroquinolone can be quickly judged by comparing the melting curves of the sample to be detected and the positive quality control product:
judging whether the sample to be detected is a mycobacterium tuberculosis complex or not by comparing the difference of the CY5 channel melting temperature (namely Tm value) obtained by the melting curves of the sample to be detected and the positive quality control product: when the Tm value of the sample to be tested is consistent with that of the positive quality control product or any Tm value (the error is less than or equal to 1 ℃) of 57.3 ℃ and 60.8 ℃ exists, the tuberculosis is judged to be positive; judging the sample to be tested CY5 as a non-tuberculosis sample if the channel has no peak or the Tm value difference between the channel and the positive control is more than or equal to 2 ℃;
when the sample CY5 to be tested is judged to be combined positive, the sample CY5 to be tested is judged to be wild type when the Tm obtained by the melting curves between the sample to be tested and the positive quality control product in the other three channels are consistent (the error is less than or equal to 1 ℃), and the test strain is sensitive to rifampicin, isoniazid and fluoroquinolone; if the difference of Tm values between the FAM channel sample in the tube A or the tube B and the positive quality control product is more than or equal to 2 ℃, judging that the rifampicin is resistant; if the difference of Tm values between the ROX channel sample in the tube A and the positive quality control product is more than or equal to 2 ℃, determining that the isoniazid is resistant; if the difference of Tm values between the VIC channel in the tube A or the tube B and the ROX channel sample in the tube B and the positive quality control material is more than or equal to 2 ℃, the fluoroquinolone medicine resistance is judged.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention develops a method for rapidly identifying mycobacterium tuberculosis complex by taking a probe melting curve technology as a basis, and can simultaneously judge the drug resistance of the mycobacterium tuberculosis complex to isoniazid, rifampicin and fluoroquinolone drugs.
(2) The insertion sequence IS6110 IS a conserved fragment of multiple copies in the genome of Mycobacterium tuberculosis and IS only present in the Mycobacterium tuberculosis complex. The sequence has high copy number (about 10-25) in most mycobacterium tuberculosis isolates, IS6110 IS selected as a positive and negative judgment target of the mycobacterium tuberculosis complex, and the detection sensitivity IS obviously improved.
(3) The invention selects rifampicin (rpoB), isoniazid (katG, inhA) and fluoroquinolone (gyrA) drug-resistant high-frequency mutation decision regions to design primers and probes, thereby being capable of rapidly judging whether a sample to be detected is multi-drug resistant/widely drug resistant.
(4) The multiple primer system is easy to cause unsatisfactory amplification results due to primer dimer, non-specific amplification and the like, is reasonable in arrangement, is suitable for amplification in the same system under the same condition, and has the advantages of high detection sensitivity, high accuracy and the like.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:
FIG. 1 is a graph showing the results of melting curves of CY5 channel in reaction systems A and B in example 3;
FIG. 2 is a graph showing the results of the melting curve of the FAM channel in the reaction system A in example 3;
FIG. 3 is a graph showing the results of the HEX channel melting curve in the reaction system A in example 3;
FIG. 4 is a graph showing the results of a melting curve of ROX channel in the reaction system A in example 3;
FIG. 5 is a graph showing the results of the melting curve of the FAM channel in the reaction system B in example 3;
FIG. 6 is a graph showing the results of the HEX channel melting curve in the reaction system B in example 3;
FIG. 7 is a graph showing the results of the melting curve of the ROX channel in the reaction system B of example 3.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
Example 1 provides primers and probes
The present example provides primer pairs for amplifying specific genes IS6110 and MPB64 of Mycobacterium tuberculosis, and corresponding probes for determining whether the specific genes are Mycobacterium tuberculosis complex flora. In addition, a primer pair and a probe are respectively designed according to rifampicin, isoniazid and fluoroquinolone drug-resistant genes and are used for detecting rpoB (codon 513 of 507-), gyrA (codon 67-106), katG315 site and inhA (-codon 17-8).
The 5 'end of the probe is marked with a fluorescent group (FAM, VIC, ROX or CY5), and the 3' end is marked with a quenching group BHQ or DABCYL. Since rpoB and gyrA drug-resistant genes are long and one probe cannot completely cover, a plurality of probes are provided. The primer pairs and probes provided in this example were aligned for homology by BLAST to ensure specificity. Primers and probes were synthesized by Guangzhou Jinzhi Biotechnology, Inc. The specific sequences of the primers and probes of the detection system for Mycobacterium tuberculosis complex provided in this example are shown in Table 1.
TABLE 1 primer and Probe sequences
Example 2 DNA extraction of test samples
This embodiment regards the sputum sample as the sample that awaits measuring, carries out DNA extraction, includes following step:
(1) adding 3 times of 4% NaOH solution into the sputum sample; vortex, shake and mix evenly, put for 15min at room temperature, then absorb 1mL and add into centrifuge tube with screw cap;
(2) centrifuging the centrifugal tube in the step (1) at 12000rpm for 5min, and removing supernatant; adding 1mL of 0.9% NaCl solution, mixing, centrifuging at 12000rpm for 5min, and discarding the supernatant.
(3) Adding 100 mu L of DNA extracting solution into the precipitate obtained in the step (2); heating at 100 deg.C for 10min, and rapidly cooling (in a refrigerator at-20 deg.C) for 10 min.
(4) And (4) centrifuging the centrifuge tube cooled in the step (3) at 12000rpm for 2min, and transferring the supernatant into a new centrifuge tube for later use, so as to prepare the template used in the subsequent detection.
EXAMPLE 3 preparation and detection of reaction solution
Two reaction systems are prepared for multiple multicolor detection:
reaction system A: 1 XPCR buffer ((NH)4)2SO4 10mM、MgCl22.5mM, KCl 10mM, Tris-HCl 20mM at pH 8.3), dNTPs 0.2mM, F-IS6110, R-IS6110, F-MPB64, R-MPB64, F-rpoB, R-rpoB, F-gyrA, R-gyrA, F-katG, R-katG, F-hA, R-inhA each 0.2. mu. mol/L, P-IS6110, P-MPB64, P1-rpoB, P3-rpoB, P2-gyrA, P4-gyrA, P-katG, P-inhA each 0.2. mu. mol/L, 2U heat-start DNase (0.3. mu.L), ddH2O is prepared to the total volume of 20 mu L; add 5. mu.L of template to be tested.
Reaction system B: 1 XPCR buffer ((NH)4)2SO4 10mM、MgCl2 2.5mM、KCTris-HCl 20mM at pH 8.3, 10 mM), dNTPs 0.2mM, F-IS6110, R-IS6110, F-MPB64, R-MPB64, F-rpoB, R-rpoB, F-gyrA, R-gyrA each 0.2. mu. mol/L, P-IS6110, P-MPB64, P2-rpoB, P4-rpoB, P1-gyrA, P3-gyrA, P5-gyrA each 0.2. mu. mol/L, 2U of heat-start DNase (0.3. mu.L), ddH 20mM), dNTPs2O is prepared to the total volume of 20 mu L; add 5. mu.L of template to be tested.
A wild-type (drug-sensitive) standard plasmid was used as a positive control at a concentration of 100 copies/. mu.L, and the DNA extract described in step (3) of example 2 was used as a negative control.
The PCR reaction program is:
the first step is as follows: 10min at 95 ℃;
the second step is that: 25s at 95 ℃, 25s at 70 ℃ (1 ℃ reduction per cycle), 30s at 72 ℃, 13 cycles;
the third step: 25s at 95 ℃, 25s at 58 ℃, 30s at 72 ℃ and 42 cycles; collecting fluorescence signals at the temperature of 72 ℃ for 30 s;
the fourth step: 1min at 95 ℃ and 1min at 45 ℃;
the fifth step: collecting fluorescence signals at 45-85 ℃ every 1 ℃, wherein FAM, VIC, ROX and CY5 are selected as fluorescence channels.
Judging the result:
in the reaction system A, referring to FIG. 1, the positive control Tm values of CY5 channel Mycobacterium tuberculosis complex are two peaks at 57.3 ℃ +/-1 ℃ and 60.8 ℃ +/-1 ℃; referring to FIG. 2, the FAM channel wild type controls have two peaks with Tm values of 62.2 ℃. + -. 1 ℃ and 81.2 ℃. + -. 1 ℃; referring to FIG. 3, the Tm values of the VIC channel wild type controls are bimodal at 63.8 ℃. + -. 1 ℃ and 75.5 ℃. + -. 1 ℃; referring to FIG. 4, the ROX channel wild type control Tm values are bimodal at 64.5 ℃. + -. 1 ℃ and 74 ℃. + -. 1 ℃.
In the reaction system B, referring to FIG. 1, the positive control Tm values of the CY5 channel Mycobacterium tuberculosis complex are two peaks at 57.3 ℃ +/-1 ℃ and 60.8 ℃ +/-1 ℃; referring to FIG. 5, FAM channel wild type control Tm values are doublets of 70.3 ℃. + -. 1 ℃ and 80.4 ℃. + -. 1 ℃; referring to FIG. 6, the VIC channel wild type control Tm values are doublets of 64 ℃. + -. 1 ℃ and 73.6 ℃. + -. 1 ℃; referring to FIG. 7, the ROX channel wild type control Tm value is a single peak at 71.5 ℃. + -. 1 ℃.
Judging whether the sample is a mycobacterium tuberculosis complex or not by comparing the difference of the CY5 channel melting temperature (namely Tm value) obtained by the melting curve between the detection sample and the positive control: when the Tm values of the sample and the positive control are consistent or one Tm value (the error is less than or equal to 1 ℃) exists between 57.3 ℃ and 60.8 ℃, the tuberculosis positivity is judged; judging the sample CY5 as a non-tuberculosis sample when the channel has no peak or the Tm value difference between the sample CY5 channel and the positive control is more than or equal to 2 ℃;
when the test sample CY5 is judged to be positive in combination, the Tm obtained by the melting curves of the samples in the other three channels and the positive control are consistent (the error is less than or equal to 1 ℃), the test strain is judged to be wild type, and the test strain is sensitive to rifampicin, isoniazid and fluoroquinolone; if the Tm value difference between the FAM channel sample in the tube A or the tube B and the positive control is more than or equal to 2 ℃, judging that the rifampicin is resistant; if the Tm value difference between the ROX channel sample in the tube A and the positive control is more than or equal to 2 ℃, determining that the isoniazid is resistant; if the difference of Tm values between the VIC channel in the tube A or the tube B and the ROX channel sample in the tube B and the positive control is more than or equal to 2 ℃, the fluoroquinolone drug resistance is judged.
Example 4 kit I
This example provides a kit comprising tube A, tube B, DNA polymerase, a negative control, and a positive control.
Tube A: 1 XPCR buffer ((NH)4)2SO4 10mM、MgCl22.5mM, KCl 10mM, Tris-HCl 20mM at pH 8.3), dNTPs 0.2mM, F-IS6110, R-IS6110, F-MPB64, R-MPB64, F-rpoB, R-rpoB, F-gyrA, R-gyrA, F-katG, R-katG, F-hA, R-inhA each 0.2. mu. mol/L, P-IS6110, P-MPB64, P1-rpoB, P3-rpoB, P2-gyrA, P4-gyrA, P-katG, P-inhA each 0.2. mu. mol/L.
A tube B: 1 XPCR buffer ((NH)4)2SO4 10mM、MgCl22.5mM, KCl 10mM, Tris-HCl 20mM at pH 8.3), dNTPs 0.2mM, F-IS6110, R-IS6110, F-MPB64, R-MPB64, F-rpoB, R-rpoB, F-gyrA, R-gyrA each 0.2. mu. mol/L, P-IS6110, P-MPB64, P2-rpoB, P4-rpoB, P1-gyrA, P3-gyrA, P5-gyrA each 0.2. mu. mol/L.
The concentration of DNA polymerase was 5U/. mu.L. When used, 19.7. mu.L of the tube A solution or the tube B solution was added with 0.3. mu.L of DNA polymerase.
Wherein the positive quality control substance is wild type (drug sensitive type) standard plasmid, and the negative quality control substance is DNA extract or sterile water.
By using the kit I provided by the embodiment, the detection of the Mycobacterium tuberculosis complex flora can be performed by using DNA extracted from a sample to be detected as a template.
Example 5 kit II
The present invention provides a kit II, which is based on the kit I described in example 4, and further comprises a DNA extraction solution. The components of the DNA extracting solution comprise ethylene diamine tetraacetic acid disodium, Tris and TritonX-100.
By using the kit II provided by the embodiment, DNA can be extracted from a sample to be detected, and the extracted DNA is used as a template to detect the Mycobacterium tuberculosis complex flora.
EXAMPLE 6 sample testing
In this example, DNA extraction and Mycobacterium tuberculosis complex detection (fluorescence PCR melting curve method) were performed on 500 samples to be tested using the kit II provided in example 5. The detection result shows that 306 samples to be detected are positive strains of mycobacterium tuberculosis, wherein 118 are rifampicin-resistant strains, 107 are isoniazid-resistant strains, and 85 are fluoroquinolone-resistant strains. Comparing the detection result of the embodiment with the clinical drug sensitivity result, the total accuracy of the kit reaches 96.08%, the sensitivity reaches 96.58%, and the specificity reaches 94.25%.
The method comprises the following specific steps:
firstly, extracting DNA of a sample to be detected:
(1) adding 3 times of 4% NaOH solution into the sputum sample; vortex, shake and mix evenly, put for 15min at room temperature, then absorb 1mL and add into centrifuge tube with screw cap;
(2) centrifuging the centrifugal tube in the step (1) at 12000rpm for 5min, and removing supernatant; adding 1mL of 0.9% NaCl solution, mixing, centrifuging at 12000rpm for 5min, and discarding the supernatant.
(3) Adding 100 mu L of DNA extracting solution into the precipitate obtained in the step (2); heating at 100 deg.C for 10min, and rapidly cooling (in a refrigerator at-20 deg.C) for 10 min.
(4) And (4) centrifuging the centrifuge tube cooled in the step (3) at 12000rpm for 2min, and transferring the supernatant into a new centrifuge tube for later use, so as to prepare the template used in the subsequent detection.
(II) preparing a reaction system:
reaction System A
| Reagent | Dosage of |
| Tube A solution | 19.7μL |
| DNA polymerase | 0.3μL |
| Form panel | 5μL |
Reaction System B
Positive control: taking a positive quality control product as a template; negative control: and taking the negative quality control product as a template.
(III) PCR amplification and detection:
the first step is as follows: 10min at 95 ℃;
the second step: 25s at 95 ℃, 25s at 70 ℃ (1 ℃ reduction per cycle), 30s at 72 ℃, 13 cycles;
the third step: collecting fluorescence signals at 95 ℃ for 25s, 58 ℃ for 25s, 72 ℃ for 30s, 42 cycles, and 72 ℃ for 30 s;
the fourth step: 1min at 95 ℃ and 1min at 45 ℃;
the fifth step: collecting fluorescence signals at 45-85 ℃ every 1 ℃, wherein FAM, VIC, ROX and CY5 are selected as fluorescence channels.
(IV) kit reference values:
the positive control Tm value of the CY5 channel Mycobacterium tuberculosis complex in the tube A is two peaks at 57.3 +/-1 ℃ and 60.8 +/-1 ℃; the FAM channel wild type contrast Tm value is a double peak of 62.2 ℃ +/-1 ℃ and 81.2 ℃ +/-1 ℃; the contrast Tm values of the wild type of the VIC channel are two peaks at 63.8 +/-1 ℃ and 75.5 +/-1 ℃; the ROX channel wild type control Tm values are bimodal at 64.5 ℃. + -. 1 ℃ and 74 ℃. + -. 1 ℃.
The positive control Tm values of the CY5 channel Mycobacterium tuberculosis complex in the tube B are double peaks at 57.3 +/-1 ℃ and 60.8 +/-1 ℃; the FAM channel wild type contrast Tm value is 70.3 +/-1 ℃ and 80.4 +/-1 ℃ double peaks; the contrast Tm values of the wild type of the VIC channel are double peaks at 64 +/-1 ℃ and 73.6 +/-1 ℃; the ROX channel wild type control Tm value is 71.5 ℃ +/-1 ℃ single peak.
(V) interpretation of results:
judging whether the sample is a mycobacterium tuberculosis complex or not by comparing the difference of the CY5 channel melting temperature (namely Tm value) obtained by the melting curves of the detection sample and the positive control: when the Tm value between the sample and the positive control is consistent or only a single Tm value (the error is less than or equal to 1 ℃) at 60.8 ℃, the positive mycobacterium tuberculosis is judged; judging that the sample CY5 has no peak or the Tm value difference between the sample CY5 channel and the positive control is more than or equal to 2 ℃ to be a non-tuberculosis mycobacterium sample;
when the test sample CY5 is judged to be positive in combination, the Tm obtained by the melting curves of the samples and the positive control in the other three channels is uniform (the error is less than or equal to 1 ℃), the test strain is judged to be wild type, and the test strain is sensitive to rifampicin, isoniazid and fluoroquinolone; if the Tm value difference between the FAM channel sample in the tube A or the tube B and the positive control is more than or equal to 2 ℃, judging that the rifampicin is resistant; if the Tm value difference between the ROX channel sample in the tube A and the positive control is more than or equal to 2 ℃, determining that the isoniazid is resistant; if the difference of Tm values between the VIC channel in the tube A or the tube B and the ROX channel sample in the tube B and the positive control is more than or equal to 2 ℃, the fluoroquinolone drug resistance is judged.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Sequence listing
<110> Guangzhou Diao Biotech Co., Ltd
<120> method and kit for detecting mycobacterium tuberculosis complex flora based on melting curve
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctcagcggat tcttcggtcg tggtc 25
<210> 2
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgaggtcgcc cgtctacttg gtgtt 25
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acatcagcct gcccagttac taccc 25
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcagcctgcc acagcgtgtc ata 23
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tggaggcgat cacaccgcag ac 22
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gtgcacgtcg cggacctcca gcc 23
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tggagcagat gggcttggg 19
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccagcagggc tcttcgtca 19
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cggaaatcgc agccacgtta c 21
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
acgggatacg aatgggggtt tg 22
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
gcaatgttcg attccggctt cc 22
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cgggcttcgg tgtacctcat 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cggcgcaccc acttacgccg 20
<210> 14
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ctggaaaatt acatcgccca g 21
<210> 15
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cattggcacc agccagctga gccaatg 27
<210> 16
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cctgcttcat ggaccagaac aacccgctgg cagg 34
<210> 17
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
cctgctcggg gttgacccac aagcgcgcag g 31
<210> 18
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ccatggcgac tgtcggcgct gccatgg 27
<210> 19
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cctgcggacg cgatcaccag cggcagcagg 30
<210> 20
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cctgccccga caacctatcg tctcgccgca gg 32
<210> 21
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cgaccgcagc cacgccaagt cggtc 25
<210> 22
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ccccgttcag ttgccgagac catgggg 27
<210> 23
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
cctgccaact accacccgca cggcgacgcg gcagg 35
<210> 24
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cctgctcgat ctacgacagc ctggtgcggc agg 33
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
catggccctg ccctggtcgc tgcgccatg 29
Claims (10)
1. The multiplex primer for detecting the mycobacterium tuberculosis complex IS characterized by comprising a specific primer pair with an insertion sequence IS6110, an MPB64 gene, an rpoB gene, a katG gene, an inhA gene and a gyrA gene; the sequence of the specific primer pair is as follows:
F-IS6110：CTCAGCGGATTCTTCGGTCGTGGTC(SEQ ID NO：1)；
R-IS6110：TGAGGTCGCCCGTCTACTTGGTGTT(SEQ ID NO：2)；
F-MPB64：ACATCAGCCTGCCCAGTTACTACCC(SEQ ID NO：3)；
R-MPB64：TCAGCCTGCCACAGCGTGTCATA(SEQ ID NO：4)；
F-rpoB：TGGAGGCGATCACACCGCAGAC(SEQ ID NO：5)；
R-rpoB：GTGCACGTCGCGGACCTCCAGCC(SEQ ID NO：6)；
F-katG：TGGAGCAGATGGGCTTGGG(SEQ ID NO：7)；
R-katG：CCAGCAGGGCTCTTCGTCA(SEQ ID NO：8)；
F-inhA：CGGAAATCGCAGCCACGTTAC(SEQ ID NO：9)；
R-inhA：ACGGGATACGAATGGGGGTTTG(SEQ ID NO：10)；
F-gyrA：GCAATGTTCGATTCCGGCTTCC(SEQ ID NO：11)；
R-gyrA：CGGGCTTCGGTGTACCTCAT(SEQ ID NO：12)。
2. the multiplex primer of claim 1, further comprising probes specific for the insertion sequences IS6110, MPB64 gene, rpoB gene, katG gene, inhA gene, and gyrA gene;
preferably, the sequence of the specific probe is:
P-IS6110：CGGCGCACCCACTTACGCCG(SEQ ID NO：13)；
P-MPB64：CTGGAAAATTACATCGCCCAG(SEQ ID NO：14)；
P1-rpoB：CATTGGCACCAGCCAGCTGAGCCAATG(SEQ ID NO：15)；
P2-rpoB：CCTGCTTCATGGACCAGAACAACCCGCTGGCAGG(SEQ ID NO：16)；
P3-rpoB：CCTGCTCGGGGTTGACCCACAAGCGCGCAGG(SEQ ID NO：17)；
P4-rpoB：CCATGGCGACTGTCGGCGCTGCCATGG(SEQ ID NO：18)；
P-katG：CCTGCGGACGCGATCACCAGCGGCAGCAGG(SEQ ID NO：19)；
P-inhA：CCTGCCCCGACAACCTATCGTCTCGCCGCAGG(SEQ ID NO：20)；
P1-gyrA：CGACCGCAGCCACGCCAAGTCGGTC(SEQ ID NO：21)；
P2-gyrA：CCCCGTTCAGTTGCCGAGACCATGGGG(SEQ ID NO：22)；
P3-gyrA：CCTGCCAACTACCACCCGCACGGCGACGCGGCAGG(SEQ ID NO：23)；
P4-gyrA：CCTGCTCGATCTACGACAGCCTGGTGCGGCAGG(SEQ ID NO：24)；
P5-gyrA：CATGGCCCTGCCCTGGTCGCTGCGCCATG(SEQ ID NO：25)。
3. the multiplex primer of claim 2, wherein the 5' end of the specific probe is modified with a fluorescent group selected from the group consisting of FAM, HEX, ROX, CY5, CY3, TET, JOE, VIC; the 3' end of the specific probe is modified with a quenching group selected from BHQ, Dabcy1, TAMRA and MGB.
4. The multiplex primer of claim 2, wherein said specific probe comprises one or both of the following modifications: modified to Peptide Nucleic Acids (PNA), Locked Nucleic Acids (LNA);
preferably, in the following specific probes, the underlined base modifications are Locked Nucleic Acids (LNAs):
P1-rpoB：CATTGGCACCAGCCAGCTGAGCCAATG；
P2-rpoB：CCTGCTTCATGGACCAGAACAACCCGCTGGCAGG；
P-inhA：CCTGCCCCGACAACCTATCGTCTCGCCGCAGG；
P2-gyrA：CCCCGTTCAGTTGCCGAGACCATGGGG。
5. use of the multiplex primer of any one of claims 1 to 4 in (i) or (ii) below:
(i) the application in detecting the mycobacterium tuberculosis complex flora comprises the steps of taking DNA of a sample to be detected as a template, and utilizing the multiplex primer to carry out amplification and melting curve detection;
(ii) application in preparing reagent for detecting Mycobacterium tuberculosis complex flora.
6. A kit for detecting Mycobacterium tuberculosis complex flora is characterized by comprising dNTPs, DNA polymerase and (NH)4)2SO4、MgCl2KCl, Tris-HCl, the multiplex primer of any one of claims 1-4;
preferably, the kit comprises a system a, a system B and a DNA polymerase, the system a comprising: dNTPs, (NH)4)2SO4、MgCl2、KCl、Tris-HCl、F-IS6110、R-IS6110、F-MPB64、R-MPB64、F-rpoB、R-rpoB、F-katG、R-katG、F-inhA、R-inhA、F-gyrA、R-gyrA、P-IS6110、P-MPB64、P1-rpoB、P3-rpoB、P-katG、P-inhA、P2-gyrA、P4-gyrA；
The system B comprises: dNTPs, (NH)4)2SO4、MgCl2、KCl、F-IS6110、R-IS6110、F-MPB64、R-MPB64、F-rpoB、R-rpoB、F-gyrA、R-gyrA、P-IS6110、P-MPB64、P2-rpoB、P4-rpoB、P1-gyrA、P3-gyrA、P5-gyrA。
7. The kit of claim 6, wherein the working concentration of the DNA polymerase is 0.05-0.2U/μ L;
preferably, the concentration of the dNTPs is 0.15-0.25 mM;
preferably, the concentration of each specific primer is independently selected from 0.02-0.5. mu. mol/L;
preferably, the concentration of each specific probe is independently selected from 0.02-0.5. mu. mol/L;
preferably, said (NH)4)2SO4In a concentration of 5-15mM, said MgCl2Is 2-5mM, the concentration of KCl is 5-15mM, and the concentration of Tris-HCl is 10-30 mM; the pH value of the Tris-HCl is 8-8.5.
8. The kit of claim 6, further comprising a negative quality control and a positive quality control;
preferably, the kit further comprises a DNA extracting solution, and the DNA extracting solution is used for extracting DNA from a sample to be detected.
9. A detection method of Mycobacterium tuberculosis complex flora is characterized by comprising the following steps:
(1) extracting DNA from a sample to be detected;
(2) performing PCR amplification and melting curve detection by using the DNA extracted in the step (1) as a template and the multiplex primer according to any one of claims 1 to 4 or the kit according to any one of claims 6 to 8;
preferably, the PCR amplification comprises the following two reaction systems:
dNTPs、(NH4)2SO4、MgCl2KCl, Tris-HCl, F-IS6110, R-IS6110, F-MPB64, R-MPB64, F-rpoB, R-rpoB, F-katG, R-katG, F-inhA, R-inhA, F-gyrA, R-gyrA, P-IS6110, P-MPB64, P1-rpoB, P3-rpoB, P-katG, P-inhA, P2-gyrA, P4-gyrA, DNA polymerase and template;
dNTPs、(NH4)2SO4、MgCl2KCl, F-IS6110, R-IS6110, F-MPB64, R-MPB64, F-rpoB, R-rpoB, F-gyrA, R-gyrA, P-IS6110, P-MPB64, P2-rpoB, P4-rpoB, P1-gyrA, P3-gyrA, P5-gyrA, DNA complex enzyme and template.
10. The method of claim 9, wherein the reaction conditions for PCR amplification and melt curve detection comprise:
S1：95℃10min；
s2: 25s at 95 ℃, 25s at 70 ℃ (1 ℃ reduction per cycle), 30s at 72 ℃, 13 cycles;
s3: collecting fluorescence signals at 95 ℃ for 25s, 58 ℃ for 25s, 72 ℃ for 30s, 42 cycles, and 72 ℃ for 30 s;
S4：95℃1min，45℃1min；
s5: fluorescence signals were collected at 45-85 ℃ every 1 ℃.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210076610 | 2022-01-24 | ||
| CN2022100766104 | 2022-01-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114480691A true CN114480691A (en) | 2022-05-13 |
Family
ID=81484847
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210198175.2A Pending CN114480691A (en) | 2022-01-24 | 2022-03-02 | Method and kit for detecting mycobacterium tuberculosis complex flora based on melting curve |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114480691A (en) |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168767A (en) * | 2006-10-25 | 2008-04-30 | 复旦大学附属华山医院 | Kit for fast detecting multiple drug resistance of mycobacteriumtuberculosis |
| CN101580879A (en) * | 2009-04-30 | 2009-11-18 | 广西医科大学 | Drug-resistance gene film chip for detecting mycobacterium tuberculosis |
| CN102229987A (en) * | 2011-05-25 | 2011-11-02 | 厦门大学 | Method and kit for detecting isoniazid resistance mutation of Mycobacterium tuberculosis |
| CN102758012A (en) * | 2012-06-15 | 2012-10-31 | 向华 | Nucleic acid fingerprint feature atlas database of mycobacterium tuberculosis rpoB gene and use of nucleic acid fingerprint feature atlas database |
| CN104561245A (en) * | 2013-10-16 | 2015-04-29 | 复旦大学 | Rapid identification method and kit for MTBC (mycobacterium tuberculosis complex) |
| CN108184327A (en) * | 2015-07-14 | 2018-06-19 | 雅培分子公司 | For identifying the composition of drug resistant M and method |
| CN109280713A (en) * | 2011-04-01 | 2019-01-29 | 澳康姆生物实验室公司 | For detecting the method and kit of cell-free pathogen specific nucleic acid |
| CN109468393A (en) * | 2017-09-06 | 2019-03-15 | 上海新培晶医学检验所有限公司 | Detect the kit and its method and purposes that MTB Quinolone Resistant Genes gyrA is mutated |
| US20190226011A1 (en) * | 2016-06-03 | 2019-07-25 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Methods for the detection of a latent tuberculosis infection |
| CN111269996A (en) * | 2020-02-10 | 2020-06-12 | 四川大学华西医院 | Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof |
| CN112501324A (en) * | 2020-11-26 | 2021-03-16 | 广州迪澳生物科技有限公司 | Primer and kit for detecting mycobacterium tuberculosis complex and nontuberculous mycobacterium complex based on loop-mediated isothermal amplification |
| CN112941210A (en) * | 2021-02-07 | 2021-06-11 | 中山大学达安基因股份有限公司 | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid |
-
2022
- 2022-03-02 CN CN202210198175.2A patent/CN114480691A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101168767A (en) * | 2006-10-25 | 2008-04-30 | 复旦大学附属华山医院 | Kit for fast detecting multiple drug resistance of mycobacteriumtuberculosis |
| CN101580879A (en) * | 2009-04-30 | 2009-11-18 | 广西医科大学 | Drug-resistance gene film chip for detecting mycobacterium tuberculosis |
| CN109280713A (en) * | 2011-04-01 | 2019-01-29 | 澳康姆生物实验室公司 | For detecting the method and kit of cell-free pathogen specific nucleic acid |
| CN102229987A (en) * | 2011-05-25 | 2011-11-02 | 厦门大学 | Method and kit for detecting isoniazid resistance mutation of Mycobacterium tuberculosis |
| CN102758012A (en) * | 2012-06-15 | 2012-10-31 | 向华 | Nucleic acid fingerprint feature atlas database of mycobacterium tuberculosis rpoB gene and use of nucleic acid fingerprint feature atlas database |
| CN104561245A (en) * | 2013-10-16 | 2015-04-29 | 复旦大学 | Rapid identification method and kit for MTBC (mycobacterium tuberculosis complex) |
| CN108184327A (en) * | 2015-07-14 | 2018-06-19 | 雅培分子公司 | For identifying the composition of drug resistant M and method |
| US20190226011A1 (en) * | 2016-06-03 | 2019-07-25 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Methods for the detection of a latent tuberculosis infection |
| CN109468393A (en) * | 2017-09-06 | 2019-03-15 | 上海新培晶医学检验所有限公司 | Detect the kit and its method and purposes that MTB Quinolone Resistant Genes gyrA is mutated |
| CN111269996A (en) * | 2020-02-10 | 2020-06-12 | 四川大学华西医院 | Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof |
| CN112501324A (en) * | 2020-11-26 | 2021-03-16 | 广州迪澳生物科技有限公司 | Primer and kit for detecting mycobacterium tuberculosis complex and nontuberculous mycobacterium complex based on loop-mediated isothermal amplification |
| CN112941210A (en) * | 2021-02-07 | 2021-06-11 | 中山大学达安基因股份有限公司 | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid |
Non-Patent Citations (2)
| Title |
|---|
| 张俊仙;王杰;孙伟民;梁艳;阳幼荣;王兰;殷铭俊;吴雪琼;: "应用荧光定量PCR-探针熔解曲线法快速鉴定分枝杆菌菌种方法的建立及初步评价", 中国医刊, no. 04, pages 74 - 78 * |
| 钟丽琪;郭亚辉;曹进;钱和;: "食源性致病菌检测技术的研究概述", 食品安全质量检测学报, no. 13, pages 257 - 263 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2011140237A2 (en) | Process for detection of multidrug resistant tuberculosis using real-time pcr and high resolution melt analysis | |
| CN111172303A (en) | Mycobacterium tuberculosis drug resistance detection kit and mycobacterium tuberculosis drug resistance detection method | |
| CN113249502A (en) | Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection | |
| CN108220444A (en) | A kind of primer combination of probe of KRAS gene mutation detection and its application | |
| Marras et al. | A molecular-beacon-based multiplex real-time PCR assay to distinguish Mycobacterium abscessus subspecies and determine macrolide susceptibility | |
| CN106498028B (en) | Diagnostic method and kit for T790M mutation of EGFR | |
| CN106498029B (en) | Method for increasing diagnostic efficiency of T790M mutation of EGFR | |
| CN108048565A (en) | A kind of primer for detecting ApoE gene pleiomorphisms and its detection method and application | |
| US20120088242A1 (en) | Assay for mycobacterium avium/intracellulare nucleic acid | |
| CN113621704B (en) | Reagent and kit for detecting and diagnosing liver cancer | |
| CN101182585A (en) | Method for identifying HBV gene mutation type, special chip and reagent kit | |
| EP1992703B1 (en) | Method for detection of mutant gene | |
| Couzinet et al. | High-density DNA probe arrays for identification of staphylococci to the species level | |
| CN104818340A (en) | Primers and kit for detecting JAK2 gene V617F site polymorphism, and PCR (polymerase chain reaction) method thereof | |
| CN116769939A (en) | Primer combination for detecting fluoroquinolone drug-resistant mutation of mycobacterium tuberculosis | |
| CN114480691A (en) | Method and kit for detecting mycobacterium tuberculosis complex flora based on melting curve | |
| CN108913774A (en) | C-KIT somatic mutation gene detection KIT and detection method thereof | |
| WO2018065830A1 (en) | Multiplex realtime pcr kit for diagnosing multidrug resistance (mdr) and extensively drug resistance (xdr) tuberculosis | |
| CN106811537A (en) | One kind detection epidermal growth factor receptor gene T790M low frequencies mutant primer and its application | |
| Wu et al. | Identification of rifampin-resistant genotypes in Mycobacterium tuberculosis by PCR-reverse dot blot hybridization | |
| Setareh et al. | High level association of mutation in KatG315 with MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus | |
| JP2005058218A (en) | Combination of circulating epstein-barr virus (ebv) dna in serum or plasma of patient and method for evaluating ebv subtype for predicting and detecting cancer related to ebv | |
| WO2020096247A1 (en) | Method for preparing probe for detecting mutation derived from cells in tissues of breast cancer and detection method | |
| WO2015083852A1 (en) | Improved cervical cancer diagnosing method and diagnostic kit for same | |
| CN116555484B (en) | EBV high-risk type fluorescent PCR detection kit |
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 |