CN107267653A - The kit and method of the rich special Salmonella of fluorogenic quantitative detection pertussis - Google Patents

The kit and method of the rich special Salmonella of fluorogenic quantitative detection pertussis Download PDF

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Publication number
CN107267653A
CN107267653A CN201710691366.1A CN201710691366A CN107267653A CN 107267653 A CN107267653 A CN 107267653A CN 201710691366 A CN201710691366 A CN 201710691366A CN 107267653 A CN107267653 A CN 107267653A
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China
Prior art keywords
reagent
pertussis
sample
detection
bordetella pertussis
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CN201710691366.1A
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Chinese (zh)
Inventor
乔阳
余倩
葛猛
王宏伟
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Beijing Fuanhua Biological Technology Co Ltd
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Beijing Fuanhua Biological Technology Co Ltd
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Priority to CN201710691366.1A priority Critical patent/CN107267653A/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/6851Quantitative amplification
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/166Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The invention discloses the kit of the rich special Salmonella of fluorogenic quantitative detection pertussis and method.The invention provides primer 1, the primer 2 of detection Bordetella pertussis, the probe 1 for detecting Bordetella pertussis of the rich special Salmonella fluorescent quantitation reagent of detection pertussis, including detection Bordetella pertussis;The experiment proves that, the present invention kit and detection method based on Taqman fluorogenic quantitative detection technologies, the pathogen that can be not only infected with fast explicit, reduce testing cost, improve equipment utilization rate, and the flux problems of clinical respiratory fungal infection pathogen detection are solved, there is important directive significance to clinical early diagnosis and therapy.

Description

The kit and method of the rich special Salmonella of fluorogenic quantitative detection pertussis
Technical field
The invention belongs to the kit in nucleic acid detection technique field, more particularly to the rich special Salmonella of fluorogenic quantitative detection pertussis And method.
Background technology
Pertussis is the Acute respiratory infectious disease as caused by Bordetella pertussis.Its Clinical symptoms is paroxysmal convulsion Contraction is coughed and with profound " crow " sample inspiratory croup, does not such as obtain timely and effective treatment, the course of disease is up to several moons.This Sick infectiousness is very strong, and children age is smaller, and the state of an illness is heavier, can because Complicating Pneumonia In Patients, encephalopathic and it is dead.
Majority of case (80-98%) is by small gramnegative bacterium Bordetella pertussis (B.pertussis) caused by, but a few cases (2-20%) are by Bordetella parapertussis (B.parapertussis) caused by (Mattoo etc.2005).By Bordetella parapertussis (B.parapertussis) symptom of pertussis caused by by Bordetella pertussis (B.pertussis) usually than being drawn That rises is gentle, however, it is difficult to be based only on clinical symptom differential diagnosis.Few is the symptom of similar pertussis, It is infected by Bordetella bronchiseptica (B.bronchiseptica) or Huo Shi Bordetellas (B.holmesii) Caused.
That circulates on the market detects that the kit of Bordetella pertussis is typically used based on fluorescent quantitation method IS481, ptxS1 gene order are as target sequence, but IS481 gene orders are present in Bordetella pertussis, suddenly simultaneously In family name's Bordetella and the rich special Salmonella of bronchus sepsis (Karen B etc.2006), ptxS1 gene orders are present in simultaneously In Bordetella pertussis and Bordetella parapertussis (Fengxiang Gao etc.2013), so these kits Can not single-minded detection Bordetella pertussis.Someone combines to detect pertussis using IS481 and IS1001 gene orders Bordetella and Bordetella parapertussis, but the combination needs two reactions to complete detection, has so aggravated detection Cost.
The content of the invention
It is an object of the present invention to provide the rich special Salmonella fluorescent quantitation reagent of detection pertussis.
The reagent that the present invention is provided, includes primer 1, the detection Bordetella pertussis of detection Bordetella pertussis Primer 2, detect Bordetella pertussis probe 1;
The nucleotides sequence of the primer 1 is classified as sequence 2;
The nucleotides sequence of the primer 2 is classified as sequence 3;
The nucleotides sequence of the probe 1 is classified as sequence 4.
In mentioned reagent, the reagent also includes internal standard fluorescence probe 2, the nucleotide sequence of the internal standard fluorescence probe 2 For sequence 6.
In mentioned reagent, the two ends difference mark fluorescent group and quenching group of each probe.
In mentioned reagent, the primer 1, the primer 2, the mol ratio of the probe 1 and the probe 2 are 2:2:1:1.
Second purpose of the invention is to provide the rich special Salmonella quantitative fluorescent PCR reagent of detection pertussis.
The PCR reagent that the present invention is provided, including mentioned reagent;
Concentration of each primer in the PCR reagent is 0.4uM;
Concentration of each probe in the PCR reagent is 0.2uM.
Kit containing mentioned reagent or above-mentioned PCR reagent is also the scope of protection of the invention.
Standard items, internal standard, positive reference substance, the negative controls of Bordetella pertussis can also be included in kit Composition.
Bordetella pertussis standard items are by the Bordetella pertussis sequence shown in sequence 1 and pGEM-T carriers (Promega, production code member A362A) is connected, and obtains Bordetella pertussis plasmid, as standard items.
Internal standard:For the interior label sequence shown in sequence 5 is connected with pGEM-T carriers, internal standard plasmid, as internal standard are obtained.
Positive reference substance wins the DNA extracts of special Salmonella for the pertussis of inactivation;
Negative controls are the sputum extract solution of normal person (extracting method is the same).
Mentioned reagent or above-mentioned PCR reagent or above-mentioned kit are in detection or quantitatively detection Bordetella pertussis Application be also the scope of protection of the invention;
Or mentioned reagent or above-mentioned PCR reagent or above-mentioned kit are preparing detection or quantitatively detection pertussis Boulder is special Application in Salmonella product is also the scope of protection of the invention;
Or mentioned reagent or above-mentioned PCR reagent or above-mentioned kit are preparing whether detection sample to be tested infects pertussis Application in Bordetella product is also the scope of protection of the invention.
To quantitative detection, it can be used in combination by detecting the Ct value sizes of 5 concentration gradient qualitative reference product (standard items) The standard curve that the Ct values size of 5 concentration gradient qualitative reference product (standard items) and 5 concentration gradients are drawn, will treat test sample This ct values are brought into the standard curve, obtain the quantitative testing result of sample to be tested;
3rd purpose of the invention is to provide a kind of method whether detection sample to be tested infects Bordetella pertussis.
The method that the present invention is provided, comprises the following steps:Real time fluorescent quantitative is carried out to sample to be tested with above-mentioned PCR reagent PCR,
If the real-time fluorescence quantitative PCR result of sample to be tested is following condition:Determining the logical of Bordetella pertussis Amplification curve is S-type under road or Ct value≤39 and Ct values > 0, then sample to be tested infection or candidate's infection pertussis Bordetella Bacterium;
If the real-time fluorescence quantitative PCR result of sample to be tested be the condition, sample to be tested do not infect or candidate not Infect Bordetella pertussis.
In the above method, the template of the PCR amplifications is the DNA of sample to be tested,
The DNA of sample to be tested and internal standard can also be mixed and be used as template by the method for the PCR amplifications.
Internal standard:For the interior label sequence shown in sequence 5 and pGEM-T carriers (Promega, production code member A362A) are connected, Obtain internal standard plasmid, as standard items.
The present invention is by Bordetella pertussis, Bordetella parapertussis, Huo Shi Bordetellas and branch gas The rich special Salmonella of pipe sepsis carries out the distinguished sequence for one section of 187bp size that sequence analysis is obtained, and the sequence exists only in pertussis In Bordetella.The kit proposed can delicately detect and it is single-minded discriminating sample present in pertussis Bordetella Sclerotium acid, has important application value in fields such as disease surveillance, clinical diagnosises.
The experiment proves that, kit of the invention and detection method based on Taqman fluorogenic quantitative detection skills Art, the pathogen that can be not only infected with fast explicit reduces testing cost, improves equipment utilization rate, and solves clinic and exhales The flux problems of road fungal infection pathogen detection are inhaled, there is important directive significance to clinical early diagnosis and therapy.
Brief description of the drawings
Fig. 1 is specificity experiments result figure of the present invention.
Fig. 2 is sensitivity test result figure of the present invention.
Fig. 3 is that clinical sample of the present invention assesses experimental result picture.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The rich special Salmonella real time fluorescent quantitative detection kit of embodiment 1, pertussis and its primer special
First, the rich special Salmonella real time fluorescent quantitative of pertussis detects the design and synthesis of primer special
By to Bordetella pertussis, Bordetella parapertussis, Huo Shi Bordetellas and bronchus sepsis Rich spy's Salmonella gene order, which is compared, filters out one section of special conserved sequence of 187bp Bordetella pertussis, the sequence It is classified as sequence 1.
Design primer sequence as follows:
Bordetella pertussis upstream amplification primer sequence is:5'-TTTCACTATGGGCTGTCGTG-3'(sequences 2);
Bordetella pertussis downstream amplification primer sequence is 5'-TGCGCTATTGCCGGATT-3'(sequences 3);
Bordetella pertussis fluorescence probe sequence is:5'-AATCCGGCAATAGCGCA-3'(sequences 4), its 5' end Flag F AM fluorophors, 3 ' end mark TAMRA quenching groups.
The present invention set in be designated as one section except probe binding sequence is different from Bordetella pertussis distinguished sequence Outside, the nucleotide sequence of other base all sames, the interior label sequence is sequence 5.
Internal standard fluorescence probe sequence is:5'-ACCTTTGGCCTGGAAACCTGGG-3'(sequences 6), internal standard probe 5' ends mark Remember JOE fluorophors, 3 ' end mark TAMRA quenching groups.
Sequence 2 and the primer of sequence 3 can be used in internal standard.
Synthesize above-mentioned primer and probe.
2nd, the foundation of the real-time fluorescence quantitative PCR detection method of the rich special Salmonella of pertussis
1st, the acquisition of the genomic DNA of sample to be tested
50 μ l samples to be tested are taken in the centrifuge tube of sterilizing, 50 μ l Lysis Buffer are added into sample to be tested, and (treasured is raw Thing engineering (Dalian) Co., Ltd, Code No.9164) and 10ul inner mark solutions (inner mark solution is that internal standard plasmid is soluble in water The solution arrived;Internal standard plasmid is in the nucleotides shown in pGEM-T carriers insetion sequence 5), it is well mixed.
80 DEG C of thermal denaturations are after 15 minutes, and low-speed centrifugal takes supernatant in the centrifuge tube of new sterilizing, obtains nucleic acid, be used for Subsequent experimental or -20 DEG C freeze.
2nd, real-time fluorescence quantitative PCR is detected
The DNA molecular of 5ul samples to be tested is added in 15ul PCR reaction solutions.
15ul PCR reaction solutions:Contain Bordetella pertussis upstream amplification primer, Bordetella pertussis downstream Amplimer be 0.4uM, Bordetella pertussis fluorescence probe and internal standard fluorescence probe concentration be 0.20uM, it is heat-resisting Archaeal dna polymerase (green skies biotech company, production code member D7207) reacts for 5U/, MgCl2Final concentration of 2.0mM, dNTPs The final concentration of 60mM of final concentration of 0.2mM, potassium chloride, 0.2% Qula lead to solution (sigma, production code member 303135, volume Percentage composition) and 10% formamide solution (sigma, production code member F9037, volumn concentration) and pH 7.5,20mM Tri(Hydroxymethyl) Amino Methane Hydrochloride (sigma, production code member 10812846001).
Above-mentioned reaction system is reacted under following response procedures:
Response procedures
FAM and JOE passages are detected at 60 DEG C respectively.In wherein FAM Air conduct measurements Bordetella pertussis, JOE detections Mark.
After reaction terminates, instrument automatically saves result, it is possible to use the software that instrument is carried is automatically analyzed, can also Initial value, end value and the threshold value of regulation baseline are analyzed manually, then record sample Ct values and result.Amplification curve with (i.e. cycle threshold refer to the threshold value that the fluorescence signal in PCR reaction tubes reaches setting for the intersection point of threshold line, referred to as Ct values When the cycling numerical value that is undergone);
If sample to be tested is following condition:It is S-type for determining FAM channel C t value≤39 (Ct values > 0) or amplification curve, Then sample to be tested infection or candidate infect Bordetella pertussis;
If sample to be tested is not above-mentioned condition:It is in S for determining FAM channel C t value≤39 (Ct values > 0) or amplification curve Type, then sample to be tested does not infect or candidate does not infect Bordetella pertussis.
The sample without Ct values is shown for determining, while internal standard test positive (Ct value≤39), is reported as feminine gender, now Sample to be tested amplification curve is straight;
For determining Ct values>39 sample, while internal standard test positive (Ct value≤39), is reported as less than under detection Limit.
If internal standard Ct values>39 or internal standard show no Ct values, then the testing result of the sample is invalid, should search and exclude original Cause, and this sample is carried out to repeat experiment.
To quantitative detection, it can be used in combination by detecting the Ct value sizes of 5 concentration gradient qualitative reference product (standard items) The standard curve that the Ct values size of 5 concentration gradient qualitative reference product (standard items) and 5 concentration gradients are drawn, will treat test sample This ct values are brought into the standard curve, obtain the quantitative testing result of sample to be tested;
Can also instrument software according to each sample Ct value sizes, pass through the standard that 5 concentration gradient qualitative reference product are drawn Curve, the quantitative testing result of each sample can be tried to achieve automatically.
3rd, the real-time fluorescence quantitative PCR detection kit of the rich special Salmonella of pertussis is stood
The real-time fluorescence quantitative PCR detection kit of the rich special Salmonella of pertussis includes:Bordetella pertussis upstream is expanded Increase primer, Bordetella pertussis downstream amplification primer, Bordetella pertussis fluorescence probe, internal standard fluorescence probe;
Or, the real-time fluorescence quantitative PCR detection kit of the rich special Salmonella of pertussis includes containing Bordetella pertussis Upstream amplification primer, Bordetella pertussis downstream amplification primer, Bordetella pertussis fluorescence probe, internal standard fluorescence are visited The PCR reagent of pin;
Internal standard fluorescence probe can also not wherein be contained in kit;
Standard items, internal standard, positive reference substance, the negative controls of Bordetella pertussis can also be included in kit Composition.
Bordetella pertussis standard items are by the Bordetella pertussis sequence shown in sequence 1 and pGEM-T carriers (Promega, production code member A362A) is connected, and obtains Bordetella pertussis plasmid, as standard items.
Internal standard:For the interior label sequence shown in sequence 5 is connected with pGEM-T carriers, internal standard plasmid, as internal standard are obtained.
Positive reference substance wins the DNA extracts of special Salmonella for the pertussis of inactivation;
Negative controls are the sputum extract solution of normal person (extracting method is the same).
The application of the rich special Salmonella real time fluorescent quantitative detection kit of embodiment 2, pertussis
First, sensitivity technique
It is 10 by concentration5Copies/ul Bordetella pertussis standard items press 10 respectively5、104、103、102、 10copies/ul gradient dilutions, afterwards using it as sample to be tested DNA, according to two fluorescent quantitation side in embodiment 1 Method is detected.
As a result as shown in fig. 2, it can be seen that the kit is minimum can to detect that about 10copies/ul is copied, with very High sensitivity.
2nd, specific test
To common other lung's pathogenic bacteria Bordetella pertussis, Bordetella parapertussis, Huo Shi Bordetellas The rich special Salmonella of bacterium, bronchus sepsis, legionella pneumophilia, streptococcus pneumonia, haemophilus influenzae, mycoplasma pneumoniae (rabbit bronchus The separation identification of sepsis bordetella bacilli and some biological characteristic researches, model will space target sequence are enriched with multiplex PCR in children Streptococcus Bacterial pathogen diagnosis in application study, Deng is after hilly) etc. extract DNA as sample to be tested nucleic acid, according to two in embodiment 1 Fluorescent quantitation method detection.
As a result it is other common as shown in figure 1, only Bordetella pertussis has amplification curve in specific test experiment Lung pathogenic germ is without amplification curve.
3rd, the rich special Salmonella real time fluorescent quantitative detection of pertussis
1st, nucleic acid extraction
The sputum for having identified 28 infection Bordetella pertussis clinical cases of BJ Children's Hospital is collected, according to reality Two method for applying example 1 extracts DNA.
2nd, real time fluorescent quantitative is detected
Above-mentioned DNA is detected according to two method of embodiment 1.
Sample for determining FAM channel C t value≤39 (Ct values > 0), is reported as the Bordetella pertussis DNA positives, Now the amplification curve of sample to be tested is S-type;
The sample without Ct values is shown for determining, while internal standard test positive (Ct value≤39), is reported as feminine gender, now Sample to be tested amplification curve is straight;
For determining Ct values>39 sample, while internal standard test positive (Ct value≤39), is reported as less than under detection Limit.
If internal standard Ct values>39 or internal standard show no Ct values, then the testing result of the sample is invalid, should search and exclude original Cause, and this sample is carried out to repeat experiment.
As a result as shown in figure 3, being detected to 28 Bordetella pertussis clinical cases, wherein 27 are pertussis Bordetella is positive.
Comparative example:
First, design control primer and control probe:
According to the primer of Bordetella pertussis gene other target regions design:
Upstream primer sequence:5'-CGGTCACGTCAAGCACAA-3'
Downstream primer sequence:5'-GGAGGATACGCCGGACT-3'
Bordetella pertussis fluorescence probe sequence is:5'-ATAGAAACAGTGGCTCGATGGCGC-3', its 5' end Flag F AM fluorophors, 3 ' end mark TAMRA quenching groups.
2nd, sensitivity technique
Detected according to one method of embodiment 2, the difference is that the primer pair and probe in this comparative example are replaced with, As a result, it is 100copies/ul to compare primer and control probe detection sensitivity, less than embodiments of the invention.
3rd, Bordetella pertussis multiple real time fluorescence quantifying is detected
Detected according to three method of embodiment 2, the difference is that the primer pair and probe in this comparative example are replaced with, As a result, only 20 Bordetella pertussis positives of 28 Bordetella pertussis clinical cases, show the positive of comparative example Recall rate is well below embodiments of the invention.
Sequence table
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<120>The kit and method of the rich special Salmonella of fluorogenic quantitative detection pertussis
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acaatcacct ttggcctgga aacctgggtg attgacaggg caatccggca atagcgcagt 120
ccggcgtatc ctccgcaggg gctctgggga aaacacgcac aaaatttcgc tgaacgaaga 180
tgagg 185
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Claims (9)

1. detecting the rich special Salmonella fluorescent quantitation reagent of pertussis, include primer 1, detection one hundred days of detection Bordetella pertussis Cough the primer 2 of Bordetella, detect the probe 1 of Bordetella pertussis;
The nucleotides sequence of the primer 1 is classified as sequence 2;
The nucleotides sequence of the primer 2 is classified as sequence 3;
The nucleotides sequence of the probe 1 is classified as sequence 4.
2. reagent according to claim 1, it is characterised in that:The reagent also includes internal standard fluorescence probe 2, the internal standard The nucleotides sequence of fluorescence probe 2 is classified as sequence 6.
3. reagent according to claim 1 or 2, it is characterised in that:The two ends difference mark fluorescent base of each probe Group and quenching group.
4. according to any described reagent in claim 1-3, it is characterised in that:
The primer 1, the primer 2, the mol ratio of the probe 1 and the probe 2 are 2:2:1:1.
5. the rich special Salmonella quantitative fluorescent PCR reagent of pertussis is detected, including any reagent in claim 1-4;
Concentration of each primer in the PCR reagent is 0.4uM;
Concentration of each probe in the PCR reagent is 0.2uM.
6. the kit containing PCR reagent described in any reagent in claim 1-4 or claim 5.
7. PCR reagent described in any reagent or claim 5 or the kit described in claim 6 in claim 1-4 Or quantitatively detection Bordetella pertussis in application;
Or PCR reagent described in any reagent or claim 5 or the kit described in claim 6 in claim 1-4 Application in preparing or quantitatively detecting Bordetella pertussis product;
Or PCR reagent described in any reagent or claim 5 or the kit described in claim 6 in claim 1-4 Preparing the application during whether detection sample to be tested infects Bordetella pertussis product.
8. a kind of detect the method whether sample to be tested infects Bordetella pertussis, comprise the following steps:With claim 5 The PCR reagent carries out real-time fluorescence quantitative PCR to sample to be tested,
If the real-time fluorescence quantitative PCR result of the sample to be tested is following condition:Determining the logical of Bordetella pertussis Amplification curve is S-type under road or Ct value≤39 and Ct values > 0, then the sample to be tested infection or candidate's infection pertussis Boulder are special Salmonella;If the real-time fluorescence quantitative PCR result of the sample to be tested is not the condition, the sample to be tested does not infect or waited Bordetella pertussis is not infected in choosing.
9. method according to claim 8, it is characterised in that:The template of the PCR amplifications is the DNA of sample to be tested.
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CN107828910A (en) * 2017-11-21 2018-03-23 弗罗朗(江苏)生物科技有限公司 A kind of kit and its application method of quick single-minded detection Bordetella pertussis

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