CN102816841A - Four-color fluorescent PCR kit for detection of vibrios in water body and detection method - Google Patents
Four-color fluorescent PCR kit for detection of vibrios in water body and detection method Download PDFInfo
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- CN102816841A CN102816841A CN201210249902XA CN201210249902A CN102816841A CN 102816841 A CN102816841 A CN 102816841A CN 201210249902X A CN201210249902X A CN 201210249902XA CN 201210249902 A CN201210249902 A CN 201210249902A CN 102816841 A CN102816841 A CN 102816841A
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Abstract
The invention discloses a four-color fluorescent PCR kit for detection of vibrios in a water body and a detection method. According to the method, innovative bacterium enrichment reagent technology, DNA extraction reagent technology, specific fluorescent probe hybridization PCR detection technology and multiplex PCR technology are employed, bacterium enrichment culture of a sample is not needed, false positive interference is minimized as much as possible on the premise that high sensitivity is maintained, and four populations of two kinds of vibrios can be detected at one time. The invention mainly has the following beneficial effects: no need for bacterium enrichment of the sample in advance; high sensitivity and good specificity in detection of bacteria; reduction of the false positive incidence in conventional PCR amplification; and realization of rapid, accurate and specific detection of vibrio cholera and vibrio parahaemolyticus at one time.
Description
Technical field
The present invention relates to Vibrio detection, be specifically related to a kind of four look fluorescent PCR kit and detection methods that detect vibrios in the water body.
Background technology
The kinds of pathogenic vibrio traditional detection method is still carried out isolation identification through microorganism culturing at present, and still perfect inadequately, it need pass through a plurality of screening steps, needs multiple substratum, and is consuming time longer; Other has the method for the monoclonal antibody detection that utilizes vibrios, and this method utilizes immunological method to identify that specificity is higher after requiring the single bacterium colony of picking to increase bacterium in a large number, but two kinds of method detection required times are all longer.Aspect molecular Biological Detection, the FDA standard is still to detect a kind of kinds of pathogenic vibrio and designs the PCR program for purpose.Domestic detection reagent design PCR also just can detect 3 kinds of vibrios during detection method simultaneously, and what pay close attention to all is O1 type and O139 type vibrio cholerae and Vibrio parahaemolyticus, and test sample also mainly is to the central Vibrio detection of food.But except common vibrio cholerae and Vibrio parahaemolyticus, many countries have begun to pay close attention to other kinds of pathogenic vibrio and the existence of vibrios in environment water.For example, deleterious hydrobiont and pathogenic agent get into new environment through ship ballast water and have been confirmed as one of significant threat that global ocean faces by world's environmental protection fund (GEF)." international hygiene regulations (2005) " are with the important supervised entities of International Voyage Ship water ballast as the port; Bear the regulatory functions of water ballast being carried causal organism as inspection and quarantine department, wherein just comprising the existence of vibrio cholerae, Vibrio parahaemolyticus and other kinds of pathogenic vibrio in the water ballast.
Quadruple fluorescent PCR know-why is based on monochromatic fluorescent PCR technology; Be meant four goal gene that in same fluorescent PCR amplification test, increase simultaneously; Probe be multiple fluorescent mark such as FAM, VIC, JOE, NED, HEX etc., the fluorescent signal that every kind of fluorescently-labeled probe is produced in the pcr amplification process is different.Because this method introduced specificity amplification primer and fluorescent probe, make that the sensitivity and the specificity that detect are strengthened significantly, thereby avoided the problem of the not high and easy omission of other detection method specificitys.
Owing in water body, there is multiple vibrios, therefore, be necessary to study a kind of method, effectively the bacterial number in the enrichment water body also can detect a plurality of kinds of pathogenic vibrio simultaneously.Like this, can when accelerating detection speed, significantly improve recall rate, reduce workload and detect cost,, integrate with international method to adapt to the new form demand of sanitary inspection.
Summary of the invention
The invention provides quadruple fluorescence PCR detection reagent kit and the detection method of vibrio cholerae and Vibrio parahaemolyticus in a kind of water body; Has the bacterial number in effective enrichment water body; Increase the bacterium process before need not; And can detect the advantage of 2 kinds of kinds of pathogenic vibrio simultaneously, and can when accelerating detection speed, significantly improve recall rate, reduce workload and detect cost.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of four look fluorescent PCR kits that detect vibrios in the water body; This test kit is on the basis of vibrio cholerae, Vibrio parahaemolyticus nucleic acid sequence analysis; Choose the conservative gene sequences Design and go out a pair of Auele Specific Primer, utilize round pcr to combine the bacterium enriching method that vibrio cholerae, Vibrio parahaemolyticus are carried out rapid detection; Increase the bacterium step before this technology need not, shortened the running time greatly, reduced pollution simultaneously, have the potential using value.Specifically, this test kit comprises that concentration is Taq archaeal dna polymerase, multiple fluorescence PCR reaction mother liquor, bacterium enrichment reagent, the DNA extraction reagent of 5U/ul.Wherein the multiple fluorescence PCR reaction mother liquor contains multiple 10 * fluorescent PCR reaction buffer, O1 crowd's cholera primer, O1 crowd's cholera probe, O139 crowd's cholera primer, O139 crowd's cholera probe, Vibrio parahaemolyticus primer, Vibrio parahaemolyticus probe, Vibrio parahaemolyticus virulent strain primer, Vibrio parahaemolyticus virulent strain probe, bovine serum albumin; Multiple 10 * fluorescent PCR reaction buffer contains Tutofusin tris hydrochloric acid, Repone K, glycerine, four kinds of mononucleotide monomers, magnesium chloride; Bacterium enrichment reagent contains PEG8000, sodium-chlor; DNA extraction reagent contains Tutofusin tris hydrochloric acid, Repone K, YD 30, NP40.
Concrete bacterium enrichment reagent preparation: PEG8000 and sodium-chlor are dissolved in pure water and are made into that PEG8000 quality percentage final concentration is 20%, the sodium-chlor final concentration is the bacterium enrichment reagent of 200mM, refrigerator 4
OCPreserve.DNA extraction reagent contains: the YD 30 of the Tutofusin tris hydrochloric acid of final concentration 20mM, the 100mM Repone K of final concentration, final concentration 2.5mM, the NP40 of quality percentage final concentration 1%, surplus is a ultrapure water, refrigerator 4
OCPreserve.Multiple 10 * PCR reaction buffer contains: the Tutofusin tris hydrochloric acid of final concentration 100mM; The Repone K of final concentration 500mM; The glycerine of quality percentage final concentration 50%; Final concentration respectively is dATP, dGTP, dTTP and the dGTP (four kinds of mononucleotide monomers) of 0.2mM, the magnesium chloride of final concentration 2mM, and surplus is a ultrapure water; The multiple fluorescence PCR reaction mother liquor is prepared by following volume ratio: multiple 10 * PCR reaction buffer 2.5ul; Concentration 20mg/ml bovine serum albumin 0.5ul; Primer O1Pf, O1Pr, O139Pf, O139Pr, toxRPf, each 0.2ul of toxRPr, primer tdhPf, each 0.3ul of tdhPr, probe O1Pb, O139Pb, toxRPb, each 0.4ul of tdhPb; No RNA enzyme DEPC water 18.3ul, preparation mixes the back in refrigerator-20
oC preserves.
Positive reference substance and negative control article can also be arranged, and positive reference substance is the pUCm-T cloning vector that is connected with the gene conserved sequence of O1 group cholera vibrio, O139 group cholera vibrio, Vibrio parahaemolyticus and virulent strain primer thereof; The negative control article are the DEPC water of no DNA enzyme.
Above-mentioned primer and probe are according to O1 group cholera vibrio RfbN gene conserved sequence design primer, probe:
O1Pf:?CCAGATTGTAAAGCAGGATGGA，
O1Pr:?GGTCATCTGTAAGTACAAC，
O1Pb:?FAM-CCCGGAGTTTGTAAGCCCACTACCGGG-DABCYL；
Design primer, probe according to O139 group cholera vibrio wbfR gene conserved sequence:
O139Pf:?CATACCAACGCCCTTATCCATT，
O139Pr:?GCATGACTGGCATCCCAAAAT，
O139Pb:?CY5-CGGGTGAGAAAAGACAGCAATAACACCCG-DABCYL；
Design primer, probe according to Vibrio parahaemolyticus toxR gene conserved sequence:
toxRPf:?AAGCGCCAGTAGTACCTGA，
toxRPr:?CCAATCTGACGGAACTGAGATT，
toxRPb:?ROX-CGGCAAATCGGTAGTAATAGTGCCG-DABCYL；
Design primer, probe according to Vibrio parahaemolyticus virulent strain tdh gene conserved sequence:
tdhPf:?AAACATCTGCTTTTGAGCTTCCA，
tdhPr:?CTCGAACAACAAACAATATCTCATCAG，
tdhPb:?HEX-CCGGGGTGTCCCTTTTCCTGCCCCCGG-Dabcyl。
Utilize this test kit to detect the method for vibrios in the water body, form by following step:
A, bacterium enrichment: under aseptic condition; Get water sample to be checked and above-mentioned bacterium enrichment reagent ratio thorough mixing in 100:1; Then 13000rpm is centrifugal 20 minutes; The SPSS of the mass percentage concentration 0.5% of the 0.5ml of supernatant discarded, and adding is then fully broken up throw out and is obtained the bacterium pregnant solution;
B, nucleic acid DNA extract: get 50ul bacterium pregnant solution and 50ul DNA extraction reagent in the 1.5ml centrifuge tube, and behind the vibration mixing, 96-100 ℃ of water-bath 10min, the centrifugal 5min of 13000rpm gets supernatant as dna profiling, stores for future use under-20 ℃ of environment;
C, the preparation of 25ul reaction system: get 2ulDNA template or positive control or negative control to the PCR pipe, add 22.6ul multiple fluorescence PCR reaction mother liquor and 0.4ul Taq archaeal dna polymerase (5U/ul), carry out the multiple fluorescence PCR amplification;
D, multiple fluorescence PCR reaction: on four look quantitative real time PCR Instruments, carry out; The probe in detecting pattern is set to: FAM, CY5, ROX and HEX passage are respectively applied for and detect O1 group cholera vibrio, O139 group cholera vibrio, Vibrio parahaemolyticus and Vibrio parahaemolyticus virulent strain; Reaction conditions: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 15s, 55 ℃ of annealing 40s (detection fluorescence), 72 ℃ are extended 15s, 40 circulations.After completion is set, preserve file, working procedure;
E, detected result are judged: S shape amplification curve occurs, the Ct value is positive less than 37, S shape amplification curve do not occur; Amplification curve is arranged perhaps but not S-shaped, threshold value surpasses 40 negatively, S shape amplification curve occurs, but the Ct value is greater than 37; Less than 40 is suspicious, to suspicious result, answers repeated experiments; If S shape amplification curve still appears in repeated experiments, negative control does not pollute, and can be judged as the positive.
The present invention has adopted bacterium enrichment reagent technology, DNA extraction reagent technology, specificity fluorescent probe hybridization PCR detection technique and the multiple PCR technique of innovation; Sample need not to increase bacterium and cultivates; Under the prerequisite that keeps hypersensitivity, reduce false positive as far as possible and disturb, and once can detect 4 kinds of populations of 2 kinds of vibrios.Beneficial effect of the present invention is mainly reflected in: need not to increase bacterium before the sample, the detection sensitivity of bacterium is high, specificity is good, has reduced the false positive rate of conventional pcr amplification; Can realize simultaneously quick, accurate, special detection to vibrio cholerae, Vibrio parahaemolyticus.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1
A kind of four look fluorescent PCR kits that detect kinds of pathogenic vibrio in the water body; This test kit is that the Taq archaeal dna polymerase of 5U/ul is formed by multiple fluorescence PCR reaction mother liquor, bacterium enrichment reagent (containing the PEG8000 of quality percentage final concentration 20%, the sodium-chlor of final concentration 200mM), DNA extraction reagent (including the Tutofusin tris hydrochloric acid of final concentration 20mM, the 100mM Repone K of final concentration, the YD 30 of final concentration 2.5mM, the NP40 of quality percentage final concentration 1%), concentration; Wherein the multiple fluorescence PCR reaction mother liquor by volume: multiple 10 * fluorescent PCR reaction buffer (includes the Tutofusin tris hydrochloric acid of final concentration 100mM; The Repone K of final concentration 500mM; The glycerine of quality percentage final concentration 50%; Final concentration respectively is dATP, dGTP, dTTP and the dGTP of 0.2mM, the magnesium chloride of final concentration 2mM) 2.5ul, concentration 20mg/ml bovine serum albumin 0.5ul; Primer O1Pf, O1Pr, O139Pf, O139Pr, toxRPf, each 0.2ul of toxRPr; Primer tdhPf, each 0.3ul of tdhPr, probe O1Pb, O139Pb, toxRPb, each 0.4ul of tdhPb, no RNA enzyme DEPC water 18.3ul forms.Positive reference substance can also be arranged: the pUCm-T cloning vector that is connected with the gene conserved sequence of O1 group cholera vibrio, O139 group cholera vibrio, Vibrio parahaemolyticus and Vibrio parahaemolyticus virulent strain primer; Negative control article: the DEPC water of no DNA enzyme.Above-mentioned primer and probe: vibrio cholerae and Vibrio parahaemolyticus nucleotide sequence are carried out bioinformatic analysis, use the homology of software analysis sequences such as Clustalw, design and screen corresponding primer, probe sequence, synthetic by Shanghai Ying Jun company.
Embodiment 2
1, chooses following pathogenic micro-organism, experimental group: O1 group cholera vibrio, O139 group cholera vibrio, Vibrio parahaemolyticus; Control group: vibrio alginolyticus, Vibrio mimicus, Vibrio flurialis, Vibrio vulnificus, Shigellae and Salmonellas; Above-mentioned pathogenic strains is all preserved from the environmental samples separation and Culture: O1 group cholera vibrio, O139 group cholera vibrio, Vibrio parahaemolyticus, vibrio alginolyticus, Vibrio mimicus, Vibrio flurialis, Vibrio vulnificus, Shigellae and Salmonellas; By comrades such as this research institute Zhou Donggens; About in March, 2011, in the Ningbo City, gather in each water body, separation and Culture, telephone number is 0574-87169626.
2, simulated environment sample preparation: the concentration with 0.5ml is 1 * 10 respectively
5The tap water thorough mixing of the O1 group cholera vibrio of CFU/ml, O139 group cholera vibrio, Vibrio parahaemolyticus, vibrio alginolyticus, Vibrio mimicus, Vibrio flurialis, Vibrio vulnificus, Shigellae and Salmonellas and 500ml is processed bacterial concentration and is 10
3The simulated environment sample of CFU/ml.
3, bacterium enrichment: under aseptic condition; Get to be checked water sample 50ml and the 0.5ml bacterium enrichment reagent thorough mixing of 5 parts of collections from ship ballast water; Then 13000rpm is centrifugal 20 minutes, supernatant discarded then, and 0.5% the SPSS that adds 0.5ml is fully broken up throw out;
4, nucleic acid DNA extracts: get each simulated environment sample of 50ul and add in the 1.5ml centrifuge tube with 50ul DNA extraction reagent respectively with each part bacterium pregnant solution; Behind the vibration mixing, 96-100 ℃ of water-bath 10min, the centrifugal 5min of 13000rpm; Get supernatant as dna profiling, store for future use under-20 ℃ of environment;
5,25ul reaction system preparation: get 2ulDNA template or positive control or negative control to the PCR pipe, add 22.6ul multiple fluorescence PCR reaction mother liquor and 0.4ul Taq archaeal dna polymerase (5U/ul), carry out the multiple fluorescence PCR amplification;
6, multiple fluorescence PCR reaction: on four look quantitative real time PCR Instruments (commercially available), carry out; The probe in detecting pattern is set to Reporter Dye:FAM, CY5, ROX and HEX passage, is respectively applied for to detect O1 group cholera vibrio, O139 group cholera vibrio, Vibrio parahaemolyticus and Vibrio parahaemolyticus virulent strain; Reaction conditions: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 15s, 55 ℃ of annealing 40s (detection fluorescence), 72 ℃ are extended 15s, 40 circulations.After completion is set, preserve file, working procedure;
7, detected result determination methods: S shape amplification curve occurs, the Ct value is positive less than 37, S shape amplification curve do not occur; Amplification curve is arranged perhaps but not S-shaped, threshold value surpasses 40 negatively, S shape amplification curve occurs, but the Ct value is greater than 37; Less than 40 is suspicious, to suspicious result, answers repeated experiments; If S shape amplification curve still appears in repeated experiments, negative control does not pollute, and can be judged as the positive; The result: O1 group cholera vibrio, O139 group cholera vibrio and Vibrio parahaemolyticus detect all positive; And in the control group vibrio alginolyticus, Vibrio mimicus, Vibrio flurialis, Vibrio vulnificus, Shigellae and Salmonellas all negative, specificity is high, reaches 100CFU/ml with detection sensitivity behind the positive reference substance doubling dilution.Water sample result to be checked: 1 this detected result of increment is the S-shaped amplification curve of FAM passage, and the Ct value is between 23, for the O1 group cholera vibrio infects; 1 this detected result of increment is CY5 and the S-shaped amplification curve of ROX passage, and the Ct value is 26 and 32, is O139 group cholera vibrio and Vibrio parahaemolyticus infection; 1 this detected result of increment is the S-shaped amplification curve of HEX passage, and the Ct value is 38.6, and S shape amplification curve still appears in duplicate detection; Negative control does not pollute; Positive Vibrio parahaemolyticus virulent strain infects, and 2 parts S shape amplification curve do not occur in addition, negative.
Embodiment 3
Step 3,4,5,6,7 same way as detect among 3 parts of tap water water samples to be checked and the embodiment 2,1 part of S-shaped amplification curve of tap water water sample ROX passage to be checked as a result, and Ct value has the Vibrio parahaemolyticus infection between 34.
< 110>Ningbo Institute of Inspection and Quarantine Science Technology
< 120>a kind of four look fluorescent PCR kit and detection methods that detect vibrios in the water body
<160> 12
<170> PatentIn?version?3.5
<210>?1
<211>?22
<212>?DNA
< 213>artificial sequence
<400> 1
CCAGATTGTA AAGCAGGATG GA ?22
<210>?2
<211>?19
<212>?DNA
< 213>artificial sequence
<400>?2
GGTCATCTGT AAGTACAAC ?19
<210>3
<211>?27
<212>?DNA
< 213>artificial sequence
<400>?3
CCCGGAGTTT GTAAGCCCAC TACCGGG ?27
<210>?4
<211>?22
<212>?DNA
< 213>artificial sequence
<400>?4
CATACCAACG CCCTTATCCA TT ?22
<210>?5
<211>?21
<212>?DNA
< 213>artificial sequence
<400>?5
GCATGACTGG CATCCCAAAA T ?21
<210>?6
<211>?29
<212>?DNA
< 213>artificial sequence
<400>?6
CGGGTGAGAA AAGACAGCAA TAACACCCG ?29
<210>?7
<211>?19
<212>?DNA
< 213>artificial sequence
<400>?7
AAGCGCCAGT AGTACCTGA ?19
<210>?8
<211>?22
<212>?DNA
< 213>artificial sequence
<400>?8
CCAATCTGAC GGAACTGAGA TT ?22
<210>?9
<211>?25
<212>?DNA
< 213>artificial sequence
<400> 9
CGGCAAATCG GTAGTAATAG TGCCG ?25
<210>?10
<211>?23
<212>?DNA
< 213>artificial sequence
<400> 10
AAACATCTGC TTTTGAGCTT CCA ?23
<210>?11
<211>?27
<212>?DNA
< 213>artificial sequence
<400>?11
CTCGAACAAC AAACAATATC TCATCAG ?27
<210>?12
<211>?27
<212>?DNA
< 213>artificial sequence
<400>?12
CCGGGGTGTC CCTTTTCCTG CCCCCGG ?27
Claims (2)
1. four look fluorescent PCR kits that detect vibrios in the water body; Comprise that concentration is Taq archaeal dna polymerase, multiple fluorescence PCR reaction mother liquor and the DNA extraction reagent of 5U/ul; It is characterized in that also comprising bacterium enrichment reagent; It is that 20% PEG8000 and final concentration are the sodium-chlor of 200mM that said bacterium enrichment reagent contains quality percentage final concentration, and said DNA extraction reagent contains the Tutofusin tris hydrochloric acid of final concentration 20mM, the 100mM Repone K of final concentration, the YD 30 of final concentration 2.5mM, the NP40 of quality percentage final concentration 1%, said multiple fluorescence PCR reaction mother liquor by volume: multiple 10 * PCR reaction buffer 2.5ul; Concentration 20mg/ml bovine serum albumin 0.5ul; Primer O1Pf, O1Pr, O139Pf, O139Pr, toxRPf, each 0.2ul of toxRPr, primer tdhPf, each 0.3ul of tdhPr, probe O1Pb, O139Pb, toxRPb, each 0.4ul of tdhPb; No RNA enzyme DEPC water 18.3ul forms; Said multiple 10 * PCR reaction buffer contains the Tutofusin tris hydrochloric acid of final concentration 100mM, the Repone K of final concentration 500mM, the glycerine of quality percentage final concentration 50%; Final concentration respectively is dATP, dGTP, dTTP and the dGTP of 0.2mM; The magnesium chloride of final concentration 2mM, the nucleotides sequence of primer O1Pf are classified CCAGATTGTA AAGCAGGATG GA as, and the nucleotides sequence of primer O1Pr is classified GGTCATCTGT AAGTACAAC as; The nucleotides sequence of probe O1Pb is classified FAM-CCCGGAGTTT GTAAGCCCAC TACCGGG-DABCYL as; The nucleotides sequence of primer O139Pf is classified CATACCAACG CCCTTATCCA TT as, and the nucleotides sequence of primer O139Pr is classified GCATGACTGG CATCCCAAAA T as, and the nucleotides sequence of probe O139Pb is classified CY5-CGGGTGAGAA AAGACAGCAA TAACACCCG-DABCYL as; The nucleotides sequence of primer toxRPf is classified AAGCGCCAGT AGTACCTGA as; The nucleotides sequence of primer toxRPr is classified CCAATCTGAC GGAACTGAGA TT as, and the nucleotides sequence of probe toxRPb is classified ROX-CGGCAAATCG GTAGTAATAG TGCCG-DABCYL as, and the nucleotides sequence of primer tdhPf is classified AAACATCTGC TTTTGAGCTT CCA as; The nucleotides sequence of primer tdhPr is classified CTCGAACAAC AAACAATATC TCATCAG as, and the nucleotides sequence of probe tdhPb is classified HEX-CCGGGGTGTC CCTTTTCCTG CCCCCGG-DABCYL as.
2. the described a kind of four look fluorescent PCR kits that detect vibrios in the water body of claim 1 detect the method for vibrios in the water body, it is characterized in that step is following:
A, bacterium enrichment: under aseptic condition; Get water sample to be checked and above-mentioned bacterium enrichment reagent ratio thorough mixing in 100:1; Then 13000rpm is centrifugal 20 minutes; The SPSS of the mass percentage concentration 0.5% of the 0.5ml of supernatant discarded, and adding is then fully broken up throw out and is obtained the bacterium pregnant solution;
B, nucleic acid DNA extract: get the above-mentioned DNA extraction reagent of 50ul bacterium pregnant solution and 50ul in the 1.5ml centrifuge tube, and behind the vibration mixing, 96-100 ℃ of water-bath 10min, the centrifugal 5min of 13000rpm gets supernatant as dna profiling, stores for future use under-20 ℃ of environment;
C, the preparation of 25ul reaction system: get 2ulDNA template or positive control or negative control to the PCR pipe, add the Taq archaeal dna polymerase of 22.6ul multiple fluorescence PCR reaction mother liquor and 0.4ul concentration 5U/ul, carry out the multiple fluorescence PCR amplification;
D, multiple fluorescence PCR reaction: on four look quantitative real time PCR Instruments, carry out; The probe in detecting pattern is set to: FAM, CY5, ROX and HEX passage are respectively applied for and detect O1 group cholera vibrio, O139 group cholera vibrio, Vibrio parahaemolyticus and Vibrio parahaemolyticus virulent strain; Reaction conditions: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 15s, 55 ℃ of annealing 40s (detection fluorescence), 72 ℃ are extended 15s, 40 circulations.After completion is set, preserve file, working procedure;
E, detected result are judged: S shape amplification curve occurs, the Ct value is positive less than 37, S shape amplification curve do not occur; Amplification curve is arranged perhaps but not S-shaped, threshold value surpasses 40 negatively, S shape amplification curve occurs, but the Ct value is greater than 37; Less than 40 is suspicious, to suspicious result, answers repeated experiments; If S shape amplification curve still appears in repeated experiments, negative control does not pollute, and can be judged as the positive.
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| CN108384869A (en) * | 2018-05-18 | 2018-08-10 | 福州大学 | The multiple PCR primer group and detection method and kit of four kinds of morbid vibrios of detection simultaneously |
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| CN105400778A (en) * | 2015-09-25 | 2016-03-16 | 深圳市疾病预防控制中心 | Kit capable of detecting 10 pathogenic vibrios simultaneously and detection method |
| CN108384869A (en) * | 2018-05-18 | 2018-08-10 | 福州大学 | The multiple PCR primer group and detection method and kit of four kinds of morbid vibrios of detection simultaneously |
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