CN106399539A - Primer and kit thereof for rapid detecting drug resistance of tubercle bacillus on streptomycin - Google Patents

Primer and kit thereof for rapid detecting drug resistance of tubercle bacillus on streptomycin Download PDF

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Publication number
CN106399539A
CN106399539A CN201610934435.2A CN201610934435A CN106399539A CN 106399539 A CN106399539 A CN 106399539A CN 201610934435 A CN201610934435 A CN 201610934435A CN 106399539 A CN106399539 A CN 106399539A
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China
Prior art keywords
primer
streptomycin
drug resistance
tubercule bacillus
kit
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曹旭东
杨彩虹
陈创夫
于潞
杨敏
袁俐
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Shihezi University
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Shihezi University
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    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a primer for detecting drug resistance of tubercle bacillus on streptomycin. The primer comprises at least one group of the following primers: RpslForward: 5'-CCAACCATCCAGCAGCTGGT-3'; RpslReverse: 5'-ATCCAGCGAACCGCGGATGA-3'; rrsForward1: 5'-ACCGGCCAACTACGTGC-3'; rrsReverse1: 5'-CGACAAACCACCTACGAGC-3'; rrsForward2: 5'-GTGTGGGTTTCCTTCCTTGG-3'; rrsReverse2: 5'-TTAATCCACATGCTCCGCC-3'; rrsForward3: 5'-AGCAACGCTGCGGTGAATAC-3'; rrsReverse3: 5'-TCCCGAGGGTTAGGCCACT-3'.

Description

One kind is used for tubercule bacillus streptomycin drug resistance quick detection primer and its test kit
Technical field
The present invention relates to a kind of research and development of biological detection reagent, especially a kind of quick for tubercule bacillus streptomycin drug resistance Detection primer and its test kit, belong to technical field of molecular biology.
Background technology
Streptomycin (Streptomycin, SM) is first antibiotic finding to interact with ribosome.Nineteen forty-four, Selman Waksman finds that SM can be used for treating tuberculosis.Early in nineteen forty-six, just it has been related to tubercule bacillus and SM has been produced The report of drug resistance.The 4th whole nation tuberculosis epidemiology investigation display in 2000, is just controlled in patient and is accounted for using SM person 88.1%, China's tubercule bacillus SM resistant rate is 17.3%；The 5th whole nation tuberculosis epidemiology investigation in 2010 finds, knot Core bacillus SM resistant rate be 19.6%, tubercule bacillus are in rising trend to the resistant rate of SM, be only second to isoniazid (isoniazid, INH) drug resistance is it should cause the highest attention of people.
Therefore, a kind of primer of rapid detecting Mycobacterium tuberculosis streptomycin drug resistance and test kit are suggested.
Content of the invention
It is an object of the invention to provide a kind of be used for tubercule bacillus streptomycin drug resistance quick detection primer and its test kit.
It is an object of the invention to provide a kind of be used for tubercule bacillus streptomycin drug resistance quick detection primer, described primer includes The least one set of following primer：
RpslForward：5’—CCAACCATCCAGCAGCTGGT—3’
RpslReverse：5’—ATCCAGCGAACCGCGGATGA—3’
rrsForward1：5’—ACCGGCCAACTACGTGC—3’
rrsReverse1：5’—CGACAAACCACCTACGAGC—3’
rrsForward2：5’—GTGTGGGTTTCCTTCCTTGG—3’
rrsReverse2：5’—TTAATCCACATGCTCCGCC—3’
rrsForward3：5’—AGCAACGCTGCGGTGAATAC—3’
rrsReverse3：5’—TCCCGAGGGTTAGGCCACT—3’.
A kind of test kit for tubercule bacillus streptomycin drug resistance quick detection it is characterised in that：At least include primer
RpslForward：5’—CCAACCATCCAGCAGCTGGT—3’
RpslReverse：5’—ATCCAGCGAACCGCGGATGA—3’
Or including primer：
rrsForward1：5’—ACCGGCCAACTACGTGC—3’
rrsReverse1：5’—CGACAAACCACCTACGAGC—3’
Or including primer：
rrsForward2：5’—GTGTGGGTTTCCTTCCTTGG—3’
rrsReverse2：5’—TTAATCCACATGCTCCGCC—3’
Or including primer：
rrsForward3：5’—AGCAACGCTGCGGTGAATAC—3’
rrsReverse3：5’—TCCCGAGGGTTAGGCCACT—3’
Table 1. rule of three detects the comparison of tubercule bacillus streptomycin resistance with high-resolution melting curve
The invention still further relates to using the above-mentioned reagent made for tubercule bacillus detection of rifampin resistant quick detection primer sets Box, using high-resolution melting curve (HRM) technology, the method establishing rapid detecting Mycobacterium tuberculosis streptomycin drug resistance.
Generally believe that Mycobacterium tuberculosis drug-resistant mechanism includes the change of cell wall permeability, the regulation and control of chlG, enzyme mistake at present The factors such as work, the mutation of target gene, the one of the main reasons wherein causing Mycobacterium tuberculosis drug-resistant is gene mutation.Molecular Epidemic Learn he result of investigation to show, the about 80% visible RpsL and rrs gene mutation of resistance to streptomycin tubercule bacillus clinical separation strain.Its Middle about 52%～59% SM Resistant strain RpsL gene is undergone mutation, and mutational site is concentrated mainly on the 43rd and the 88th ammonia Base acid, the two codon sports AGG by AAG, and aminoacid sports arginine (A rg) by lysine (L ys).Also have 8%～21% SM Resistant strain rrs gene is undergone mutation, and mutation is concentrated mainly on 530 ring region and 904 bit bases.Thus may be used See, the mutation of Rpsl gene and rrs gene and tubercule bacillus streptomycin resistance have substantial connection, therefore, can be by analyzing this The abrupt climatic change major part drug resistance to streptomycin for the tubercule bacillus of two genes.
The DNA sequence in the common mutations area according to tubercule bacillus streptomycin (SM) drug resistance related gene Rpsl and rrs for the present invention Row, the primer of design detection mutation, with high-resolution melting curve (high-resolution melting curve, HRM) point Analysis method carries out the quick detection of the streptomycin resistance of bacterial strain to 49 plants of tubercule bacillus separation strains, with traditional rule of three medicine Sensitivity testss testing result is standard, evaluates high-resolution melting curve method and detects the sensitivity of streptomycin resistance, special Property, set up a kind of method of quick detection streptomycin resistance, the quick auxiliary diagnosis for resistant tuberculosis provide technical support.
Compared with prior art, to have a detection time short for the present invention, is usually no more than 3 days, accuracy rate is high, simple to operate, Expense is low, the features such as easily popularization.
Brief description
Fig. 1 is the HRM detection and analysis result of streptomycin drug resistance related gene Rpsl.
Fig. 2 is the HRM detection of streptomycin drug resistance related gene rrs.
Specific embodiment
Embodiment 1：Experimental program is as follows：
(1) with inoculating loop scraping well-grown bacterium on modified Russell medium, put into the EP pipe equipped with TEbuffer In, 85 DEG C of metal baths inactivate 45min.
(2) extract the DNA of various bacterial strains according to the description of pillar mycobacteria DNAout test kit.
(3) survey DNA concentration, according to different concentration dilution to 10ug/ml.
Reaction system：Cumulative volume 10ul (HRM Mix：5ul；MgCl2：1.2ul；H2O：2.3ul；Respectively using following upper and lower Trip primer is each：0.5ul；DNA profiling：1ul)；Upstream and downstream primer is：
RpslForward：5’—CCAACCATCCAGCAGCTGGT—3’
RpslReverse：5’—ATCCAGCGAACCGCGGATGA—3’
rrsForward1：5’—ACCGGCCAACTACGTGC—3’
rrsReverse1：5’—CGACAAACCACCTACGAGC—3’
rrsForward2：5’—GTGTGGGTTTCCTTCCTTGG—3’
rrsReverse2：5’—TTAATCCACATGCTCCGCC—3’
rrsForward3：5’—AGCAACGCTGCGGTGAATAC—3’
rrsReverse3：5’—TCCCGAGGGTTAGGCCACT—3’；
Response parameter：95 DEG C of 10min of denaturation；95 DEG C of 10s of degeneration；60 DEG C of 15s of annealing；sec target 53℃；Extend 72 DEG C of 10s, totally 50 circulations；95℃1min；40℃1min；70℃1s；95℃continuous；40℃30s；.
2 experimental results
2.1 results judge：
(1) each sample arranges 3 repetitions.
(2) when carrying out HRM analysis, test hole corresponding Cp ＜ 30.If Cp value is more than 30, dilute sample, again Tested, until the Cp value of sample controls within 0.5 less than the change of 30 and multiple holes Cp value to reduce test error.
(3) if the peak shape of 3 multiple holes of sample is identical but different from wild type, then it is judged as mutant, otherwise be then open country Raw strain.If there being the result of 2 multiple holes different from wild type, it is judged as saltant type.If the peak shape of 3 multiple holes of sample is all different, Test then to be repeated to determine.
2.2 HRM results：
Using 16 plants of tubercule bacillus streptomycin persister, with this method detect mutation positive for 13 plants.Using sensitivity 33 plants of strain, with this method detect mutation negative for 28 plants.The sensitivity of this method is 81.25%；Specificity 81.82%. Using this group primer, carry out susceptibility detection with high-resolution melting curve (HRM) technology, obtain susceptibility information required time and do not surpass Spend 3 days.

Claims (2)

1. one kind is used for tubercule bacillus streptomycin drug resistance quick detection primer, and described primer includes the least one set of following primer：
RpslForward：5’—CCAACCATCCAGCAGCTGGT—3’
RpslReverse：5’—ATCCAGCGAACCGCGGATGA—3’
rrsForward1：5’—ACCGGCCAACTACGTGC—3’
rrsReverse1：5’—CGACAAACCACCTACGAGC—3’
rrsForward2：5’—GTGTGGGTTTCCTTCCTTGG—3’
rrsReverse2：5’—TTAATCCACATGCTCCGCC—3’
rrsForward3：5’—AGCAACGCTGCGGTGAATAC—3’
rrsReverse3：5’—TCCCGAGGGTTAGGCCACT—3’.
2. a kind of test kit for tubercule bacillus streptomycin drug resistance quick detection it is characterised in that：At least including right such as will Seek the least one set in the primer described in 1.
CN201610934435.2A 2016-10-25 2016-10-25 Primer and kit thereof for rapid detecting drug resistance of tubercle bacillus on streptomycin Pending CN106399539A (en)

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Application Number Priority Date Filing Date Title
CN201610934435.2A CN106399539A (en) 2016-10-25 2016-10-25 Primer and kit thereof for rapid detecting drug resistance of tubercle bacillus on streptomycin

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004063368A1 (en) * 2003-01-14 2004-07-29 Japan Science And Technology Agency Method of detecting drug-resistance gene contained in tubercle bacillus, primer pair set for pcr, primer set for determining base sequence and reagent kit for diagnnosing drug-resistant tuberculosis
CN105368939A (en) * 2015-11-06 2016-03-02 石河子大学 Primer for tubercle bacillus rifampicin drug-resistant fast detection and kit for tubercle bacillus rifampicin drug-resistant fast detection
CN105969861A (en) * 2016-05-17 2016-09-28 石河子大学 Primers and kit for quickly detecting Mycobacterium tuberculosis isoniazide drug resistance

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004063368A1 (en) * 2003-01-14 2004-07-29 Japan Science And Technology Agency Method of detecting drug-resistance gene contained in tubercle bacillus, primer pair set for pcr, primer set for determining base sequence and reagent kit for diagnnosing drug-resistant tuberculosis
CN105368939A (en) * 2015-11-06 2016-03-02 石河子大学 Primer for tubercle bacillus rifampicin drug-resistant fast detection and kit for tubercle bacillus rifampicin drug-resistant fast detection
CN105969861A (en) * 2016-05-17 2016-09-28 石河子大学 Primers and kit for quickly detecting Mycobacterium tuberculosis isoniazide drug resistance

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Application publication date: 20170215