CN110423829A - A kind of fluorescent PCR kit detecting Bacterium diphtheriae - Google Patents
A kind of fluorescent PCR kit detecting Bacterium diphtheriae Download PDFInfo
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Classifications
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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
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention discloses a kind of fluorescent PCR kits for detecting Bacterium diphtheriae.The fluorescent PCR kit of detection Bacterium diphtheriae of the present invention includes: one group of forward primer, reverse primer and fluorescence probe designed using the adjusting gene DtxR gene of Bacterium diphtheriae as target gene；One group of forward primer, reverse primer and fluorescence probe designed using the virulence gene Tox gene of Bacterium diphtheriae as target gene.The fluorescent PCR kit of detection Bacterium diphtheriae of the present invention not only can detecte Bacterium diphtheriae, and it can detect whether this bacterial strain carries Tox virulence gene simultaneously, therefore the judgement of bacterial strain and epidemic situation is played an important role in epidemic prevention and control work.
Description
Technical field
The invention belongs to biomedicine technical fields, are related to a kind of fluorescence PCR detection reagent kit, and in particular to a kind of detection
The fluorescent PCR kit of Bacterium diphtheriae.
Background technique
Bacterium diphtheriae is that a kind of positive Grain stain, one section of thallus or both ends are expanded in rodlike, and can pass through breathing
It propagates so as to cause the diphtheroid acute infection encephalapthy agent of people in road.The position that the pathogen is often invaded is pharynx, larynx, tracheae and nose
Chamber mucous membrane can also invade eye conjunctiva, skin and mucosa.Metainfective characteristic feature is congested nose, throat etc. mucous membrane, swelling and shape
At canescence pseudomembrane, lead to respiratory disorder.Exotoxin enters blood and causes systemic toxicity profiles symptom, serious person can lead to myocarditis or
Peripheral nerve paralysis.
Although diphtheria vaccine covers extensively in the world, according to the statistics of the World Health Organization in the upper world 90
Age, the former Soviet Union have 15.7 ten thousand people to infect and cause about 5000 people dead.Still there are 7321 diphtheria cases in the whole world within 2014, and
Case is concentrated mainly on developing country.There are outburst in the India, Laos and Thailand on China periphery in recent years.
The fifties, the sixties of the China in 19th-century, annual new cases people up to ten thousand, disease incidence are up to 10/,100,000-
20/100000.But since the Ministry of Public Health in 1962 issues " immunization campaign work implementing method " after especially 1978, diphtheria morbidity
Rate and death rate rapid decrease are down to about 3/,100,000 the seventies, and the eighties, the nineties was lower than 0.01/10 ten thousand down to 1/,100,000.According to
Health ministry infectious disease statistical information and document report only have single cases report so far after 2000.But in 2010
An example has been separated without the Bacterium diphtheriae of Tox virulence gene at the canthus of a tumor patient.More and more be free of virulence
Corynebacterium diphtheriae cause the disease such as endocarditis, pyogenic arthritis of atypical symptom or caused in uncommon position
Infection.
Diphtheria toxin is a kind of virulent property, and the protein of strong antigen is non-toxic white by the invasion of β β-cory-nephage
Larynx bacillus, Tox gene integration host chromosome and become toxigenicity corynebacterium diphtheriae.Diphtheria toxin is made of two peptide chains of A, B,
A peptide chain is diphtheria toxin toxicity functional areas, inhibits the synthesis of permissive cell protein；B peptide chain has a receptor binding domain and turns
Position area, itself is non-toxic but A peptide chain can be assisted to enter in permissive cell.DtxR is that diphtheria toxin inhibits albumen, is widely present in
Poison is produced in not toxigenic bacterium strain, corynebacterium diphtheriae is adjusted in such a way that metal relies on and generates diphtheria toxin.DtxR albumen is diphtheria
Comprehensive regulatory factor of bacillus metabolism, not only adjusts the expression of diphtheria toxin, is also responsible for adjusting siderophore and other 7 kinds of promoters
Synthesis.
Since the detection kit of current mainstream is for the Bacterium diphtheriae for having virulence, without containing virulence gene
Bacterium diphtheriae often can lead to error in judgement by the judgement of mistake to be negative.For example, Jiangsu and the wound limited public affairs of biotechnology
(application number: being to be directed to used by 201610019376.6) to " the Bacterium diphtheriae fluorescence PCR detection reagent kit " of department's application
The primer of Bacterium diphtheriae virulence gene (Tox gene).
Therefore, it is necessary to develop the kit of complete detection Bacterium diphtheriae, avoid missing inspection, with for disease prevention and
Epidemic situation control.
Need detection kit a kind of quick and that its characterizing gene and virulence gene can be detected simultaneously.
Summary of the invention
The purpose of the present invention is to provide a kind of fluorescent PCR kits for detecting Bacterium diphtheriae.
The fluorescent PCR kit of detection Bacterium diphtheriae of the present invention includes: one group with Bacterium diphtheriae
Adjust forward primer, reverse primer and fluorescence probe that gene DtxR gene designs for target gene；One group with Bacterium diphtheriae
Virulence gene Tox the gene forward primer, reverse primer and the fluorescence probe that are designed for target gene.
It is according to the present invention detection Bacterium diphtheriae fluorescent PCR kit further feature, it is described with
The sequence for the forward primer that DtxR gene designs for target gene is SEQ ID NO.1, and the sequence of reverse primer is SEQ ID
NO.2, the sequence of fluorescence probe are SEQ ID NO.3.
Preferably, the fluorescent reporter group of the fluorescence probe is FAM, non-fluorescence quenching group BHQ1.
The further feature of the fluorescent PCR kit of detection Bacterium diphtheriae according to the present invention, it is described with Tox
The sequence for the forward primer that gene designs for target gene is SEQ ID NO.4, and the sequence of reverse primer is SEQ ID NO.5, glimmering
The sequence of light probe is SEQ ID NO.6.
Preferably, the fluorescent reporter group of the fluorescence probe is HEX, non-fluorescence quenching group BHQ1.
The fluorescent PCR kit of detection Bacterium diphtheriae of the present invention not only can detecte Bacterium diphtheriae,
And it can detect whether this bacterial strain carries Tox virulence gene simultaneously, therefore for bacterial strain and epidemic situation in epidemic prevention and control work
Judgement play an important role.
Firstly, the detection of this kit is Bacterium diphtheriae DtxR gene, this gene is that diphtheria toxin inhibits albumen base
Cause.Diphtheria toxin inhibits albumen to be widely present in Bacterium diphtheriae production strain and do not produce strain, in such a way that metal relies on
It adjusts Bacterium diphtheriae and generates diphtheria toxin.DtxR gene (Gene ID:29421139) overall length 681bp, it is highly conserved, because
Whether the FAM fluorescence of this this kit can detecte has dtxR base then determine whether for Bacterium diphtheriae.
Secondly, this kit can pass through the Tox virulence gene of HEX fluorescence detection Bacterium diphtheriae simultaneously.Corynebacterium diphtheriae
By Tox gene expression diphtherotoxin, exotoxin enters into the cell bacillus in conjunction with sensitive cells, inhibits intracellular protein
Matter synthesis, and cause intracellular granular object increase, followed by cell aggregation, fall off.It is filled so as to cause the positions such as human body throat mucous membrane
Simultaneously there is white pseudomembrane and human body are caused the acute upper respiratory infection diseases such as fever, cough, inspiratory dyspnea occur in blood, swelling
Shape, serious person may occur in which myocarditis, neural paralysis etc..Tox toxin is the main morbid substance of bacterial strain, without containing Tox gene
Strain Virulence is weak, therefore can speculate whether bacterial strain has high virulence to the identification of Tox virulence gene.
Traditional method for cultivation of bacteria is the goldstandard of Bacteria Identification, and the culture of Bacterium diphtheriae needs special culture
Base Lv Shi serum inclined-plane or potassium tellurite mediuin；The exotoxin that Bacterium diphtheriae generates is its main pathogenic factor, only
Exotoxin could be generated by carrying β-β-cory-nephage.Its exotoxin: Ai Li is detected usually using following two methods
Gram plate virulence experiment and cavy intradermal vaccination virulence experiment.Ai Like plate virulence experiment is needed using diphtheria antitoxin (DAT), training
Supporting the time is 72 hours；Cavy intradermal vaccination virulence experiment needs two cavys and diphtheria antitoxin (DAT), observes after needing 72 hours
Inoculation position abnormal conditions.The operation of both virulence experiment methods is complex.
Kit of the present invention can detect Bacterium diphtheriae and its virulence using double fluorescent round pcr simultaneously
Gene only need to can judge whether it is diphtheria at 1 hour using this kit for after the Nasopharyngeal swabs sample extraction nucleic acid taken
Corynebacteria and whether contain virulence gene.Kit high sensitivity of the present invention, high specificity is easy to operate, cost
Cheap, detection time is quick.
Detailed description of the invention
Fig. 1 is the sensitivity technique curve of kit detection corynebacterium diphtheriae DtxR gene of the present invention.
Fig. 2 is the sensitivity technique curve of kit detection corynebacterium diphtheriae Tox gene of the present invention.
Specific embodiment
Below by way of specific embodiment and in conjunction with attached drawing, the present invention is described in detail.
1. design of primers
The adjusting gene DtxR gene and virulence gene Tox gene for selecting Bacterium diphtheriae are as target gene design primer
With probe (being shown in Table 1).
The primer and probe sequence of table 1:DtxR gene and Tox gene
Note: the expanding fragment length of DtxR gene is 129bp, and the expanding fragment length of Tox gene is 133bp.
The fluorescence probe of DtxR gene is preferred are as follows: FAM-CGATACCACAGAGATGTACTTGCG-BHQ1.
The fluorescence probe of Tox gene is preferred are as follows: HEX-TACCGACAATAAATACGACGCTGC-BHQ1
The preparation of 2.DNA template
It is taken in 100 μ l to 1.5ml centrifuge tubes after the Nasopharyngeal swabs sample of acquisition is mixed, 50 μ l lysates of addition, 100 DEG C
Heated at constant temperature 10min, 12000rpm are centrifuged 2min, and supernatant is the template prepared.Commercialization DNA can also be used to extract reagent
Box mentions sample DNA.
3. Fluorescence PCR system and amplification program
Each sample reaction system are as follows: 5 μ l of reaction solution 20 μ l, sample to be tested DNA.
Sense channel is FAM and HEX, and it is none that channel, which is quenched,.
PCR cycle parameter setting are as follows: 95 DEG C of 10min；95 DEG C of 5sec, 55 DEG C of 30sec, 40 circulations, acquire at 55 DEG C
Fluorescence.
In test sample, positive control and negative control need to be done simultaneously.
4. result judges
The Ct value in the channel FAM and the channel HEX≤35, and has exponential growth curve, then Bacterium diphtheriae and virulence base
Because being the positive.
The channel FAM and the equal > 35 of HEX channel C t value, and have exponential growth curve, then result is suspicious, needs to repeat real
It tests.
The channel FAM and the equal > 35 of HEX channel C t value, no exponential growth curve, then result is feminine gender.
Value≤35 FAM channel C t > 35, HEX channel C t, then Bacterium diphtheriae is positive, and virulence gene is negative.
5. sensitivity experiment
Corynebacterium diphtheriae reference culture (CMCC38009) fresh colony is diluted to uniform bacteria suspension with 10mlPBS by 5.1,
And gradient dilution is at 1:10,1:102、1:103、1:104、1:105、1:106Deng the bacteria suspension of 6 dilutions.
5.2 each dilution bacterium colonies count.The 100 μ l of bacteria suspension of each dilution is taken uniformly to be applied on blood plate respectively
(each dilution does a Duplicate Samples simultaneously), then by blood plate at 37 DEG C after aerobic incubator culture 1 day, on blood plate
Corynebacterium diphtheriae bacterium colony count respectively.And the concentration of bacteria suspension is calculated referring to " GB4789.2-2006 ".
5.3 each dilutions extract nucleic acid and PCR.Take each dilution 100 μ l, 95 DEG C of heating 10min, 12000rpm from
Heart 2min, supernatant are stand-by template.The template of each dilution does fluorescent PCR according to previous reaction system.
5.4 sensitivity experiment results (being shown in Table 2)
Table 2: the sensitivity technique experimental result of diphtheria reference culture
| Dilution | Bacterial concentration (CFU/ μ l) | As a result (DtxR/Tox) |
| Stoste | 6×104 | +/+ |
| 1:101 | 6×103 | +/+ |
| 1:102 | 6×102 | +/+ |
| 1:103 | 6×101 | +/+ |
| 1:104 | 6×100 | +/+ |
| 1:105 | 6×10-1 | -/- |
| 1:106 | 6×10-2 | -/- |
Fig. 1 shows corynebacterium diphtheriae DtxR gene sensitivity technique curve.It will be seen from figure 1 that dilution arrives for stoste
1:104For 5 gradient dilution degree there is Exponential growth stage, and 1:105And 1:106Do not occur Exponential growth stage.Therefore this
For kind method to the detection limit of corynebacterium diphtheriae DtxR gene, the concentration of corresponding bacterium solution is 6CFU/ μ l.
Fig. 2 shows corynebacterium diphtheriae Tox virulence gene sensitivity technique curve.Figure it is seen that dilution is stoste
To 1:104For 5 gradient dilution degree there is Exponential growth stage, and 1:105And 1:106Do not occur Exponential growth stage.Therefore
For this method to the detection limit of corynebacterium diphtheriae Tox gene, the concentration of corresponding bacterium solution is 6CFU/ μ l.
6. specific test
6.1 bacterium sources: reference culture listed by table 3 is purchased from American type culture collection (ATCC) or middle traditional Chinese medical science
It learns bacterium preservation administrative center (CMCC), avirulence Bacterium diphtheriae is clinical separation strain.
6.2, by the bacterial strain of table 3 brain heart infusion broth recovery Zengjing Granule, take bacterium solution 1ml, 12000rpm to be centrifuged 2min,
Remove supernatant, be added 50 μ l DNA extracting solutions, 95 DEG C of 10min, 12000rpm are centrifuged 2min, and supernatant is to be checked to use mould
Plate.
6.3 take above-mentioned 5 μ l of template to carry out fluorescent PCR amplification amplification according to the reaction system and reaction condition of this kit
It the results are shown in Table 3.
Table 3: the specific test of Bacterium diphtheriae detection kit
From table 3 it can be seen that corynebacterium diphtheriae FAM and the HEX binary channels containing Tox virulence gene is the positive, and be free of
There is the corynebacterium diphtheriae FAM of virulence gene for the positive, HEX is feminine gender.Rather than the experimental result of corynebacterium diphtheriae is feminine gender.
7. conclusion
The present invention establishes a kind of PCR kit that can detect Bacterium diphtheriae and its virulence gene simultaneously, sensitivity
Up to 6CFU/ μ l, specificity is 100%, can be applied to the clinical quick and precisely detection to suspicious diphtheria bacterial strain, and can distinguish simultaneously
Whether Tox virulence gene is carried.
SEQUENCE LISTING
<110>Guangzhou Disease Prevention-Control Center
<120>a kind of fluorescent PCR kit for detecting Bacterium diphtheriae
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial synthesized
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gaggcttaca atgaagga 18
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<212> DNA
<213>artificial synthesized
<400> 2
gtaggtccag attgttcc 18
<210> 3
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<212> DNA
<213>artificial synthesized
<400> 3
cgataccaca gagatgtact tgcg 24
<210> 4
<211> 20
<212> DNA
<213>artificial synthesized
<400> 4
caaggaaatt atgacgatga 20
<210> 5
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<212> DNA
<213>artificial synthesized
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ctggatacgt cactttga 18
<210> 6
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<212> DNA
<213>artificial synthesized
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taccgacaat aaatacgacg ctgc 24
Claims (5)
1. a kind of fluorescent PCR kit for detecting Bacterium diphtheriae characterized by comprising
One group of forward primer, reverse primer and fluorescence designed using the adjusting gene DtxR gene of Bacterium diphtheriae as target gene
Probe；
One group of forward primer, reverse primer and fluorescence designed using the virulence gene Tox gene of Bacterium diphtheriae as target gene
Probe.
2. it is according to claim 1 detection Bacterium diphtheriae fluorescent PCR kit, it is characterised in that: it is described with
The sequence for the forward primer that DtxR gene designs for target gene is SEQ ID NO.1, and the sequence of reverse primer is SEQ ID
NO.2, the sequence of fluorescence probe are SEQ ID NO.3.
3. the fluorescent PCR kit of detection Bacterium diphtheriae according to claim 2, it is characterised in that: the fluorescence
The fluorescent reporter group of probe is FAM, non-fluorescence quenching group BHQ1.
4. the fluorescent PCR kit of detection Bacterium diphtheriae according to claim 1, it is characterised in that: described with Tox
The sequence for the forward primer that gene designs for target gene is SEQ ID NO.4, and the sequence of reverse primer is SEQ ID NO.5, glimmering
The sequence of light probe is SEQ ID NO.6.
5. the fluorescent PCR kit of detection Bacterium diphtheriae according to claim 4, it is characterised in that: the fluorescence
The fluorescent reporter group of probe is HEX, non-fluorescence quenching group BHQ1.
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| CN201910752525.3A CN110423829A (en) | 2019-08-15 | 2019-08-15 | A kind of fluorescent PCR kit detecting Bacterium diphtheriae |
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| CN201910752525.3A Pending CN110423829A (en) | 2019-08-15 | 2019-08-15 | A kind of fluorescent PCR kit detecting Bacterium diphtheriae |
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