CN104404144A - Kit for detecting coli groups and application - Google Patents
Kit for detecting coli groups and application Download PDFInfo
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- CN104404144A CN104404144A CN201410660228.3A CN201410660228A CN104404144A CN 104404144 A CN104404144 A CN 104404144A CN 201410660228 A CN201410660228 A CN 201410660228A CN 104404144 A CN104404144 A CN 104404144A
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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
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a kit for detecting coli groups and an application. The kit comprises a primer sequence and a probe sequence, wherein the primer sequence consists of an upstream primer F1130a, an upstream primer F1130b and a downstream primer R1264; the probe sequence is a probe Pb1156A. The kit is good in sensitivity and specificity and can replace traditional methods. The kit can be used for counting the coli groups in food or other samples quickly and effectively, manpower and material resource consumption of existing coli group detection methods can be greatly reduced, and spot check of large batches of the food or other samples in daily production and life can be simplified, so that the kit is particularly suitable for wide application in the field of the food.
Description
Technical field
The invention belongs to microorganism detection field, particularly a kind of test kit for detecting coliform and application.
Background technology
The microbial contamination of food is one of important factor affecting food safety, and in its storage with the course of processing, detecting food microbes quickly and accurately and pollute, is the important means controlling and ensure food safety.At present, for the detection of coliform in food, the detection method that domestic majority adopts is according to National Standard of the People's Republic of China " rapid detection of microbiological test of food hygiene-coliform " (GB/T4789.32-2002).Accuracy, the susceptibility of National Standard Method detected result are all higher, but trivial operations, consuming time longer, are not suitable for quality guaranteed period short food inspection; Although rapid detection National Standard Method substantially reduces the test period, operation is also relatively easy, but still needs to use a large amount of Glass Containers and instrument, is not easy to execute-in-place.Import and export 3M test paper method for quick in industry standard " in food coliform and intestinal bacteria quick counting process PetrifilmTM testing plate method " (SN/T 1896-2007) due to pre-treatment simple, treatment time is short, in the Site Detection of manufacturing and processing enterprise, there is greater advantage, but the food hygienic standard whether its Detection results and detection limitation reach national regulation need to confirm, and cost is higher, be not suitable for large batch of tainted food in daily productive life.
Due to the extensive existence of coliform, make traditional detection method become complicated and poor efficiency, be not suitable for the requirement of rapid sensitive in modern food detection.Therefore work out and a kind ofly can detect various coliform in food and universal detection method succinct is fast extremely urgent simultaneously.
Current domestic and international application, in the Protocols in Molecular Biology detecting coliform, is mainly divided three classes: regular-PCR technology, Fluorescence PCR assay and biochip technology.Method for gene chip detection efficiency is high, but technology is also immature, and false positive rate and false negative rate are all difficult to control, and cost is higher, is also in conceptual phase at present.Regular-PCR method and technology is ripe, but owing to detecting in sample, microbe species is a lot, and design of primers is difficult point, and primer specificity is not high, easily causes false positive results; And qualitative detection can only go out coliform, and can not be quantitative; Moreover need to carry out aftertreatment to PCR primer, very easily cause PCR primer to be polluted.Fluorescent PCR is on the basis of regular-PCR, a specific fluorescent probe is added again add a pair Auele Specific Primer in amplification reaction system while, the fluorescent PCR detector of Real-Time Monitoring is used to detect the technology of target nucleotide sequences, it is except the advantage with regular-PCR, also have the following advantages: (1) specificity is stronger, sensitivity is higher: due to employ more one can with the fluorescent probe of template complementary pairing, thus improve specificity, and collect fluorescent signal by self-reacting device, avoid the subjectivity of artificial judgment, can further improve sensitivity again, (2) totally-enclosed reaction, online Real-Time Monitoring fluorescence, need not the logarithmic phase of PCR, abandons the end point analysis method by multifactor interference of regular-PCR method, makes quantitatively more accurately and reliably, (3) the two inspection of single tube can be realized or examine more, also can design mark in specific aim, monitoring extraction efficiency and get rid of inhibitor interference, (4) toxic reagent is not contacted, operational safety, (5) mass-producing, automatization and network management is conducive to, (6) scope of application is wider, can detect the nucleic acid of any coliform in theory.But, quantitative fluorescent PCR for the requirement of primer relative to regular-PCR, more harsh.
Summary of the invention
Primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, provides a kind of test kit for detecting coliform.This test kit energy detection by quantitative coliform.
Another object of the present invention is to the application that described test kit is provided.
Object of the present invention is achieved through the following technical solutions: a kind of test kit for detecting coliform, comprises primer sequence and probe sequence; Wherein: primer sequence is made up of upstream primer F1130a, upstream primer F1130b and downstream primer R1264; Probe sequence is probe Pb1156A;
The sequence of described upstream primer F1130a is arbitrary sequence as described below: core sequence A; Or centered by core sequence A, hold to 5 ' and 3 ' sequence extending 1 ~ 10 Nucleotide respectively or simultaneously and obtain; The sequence be preferably core sequence A, obtaining to 5 ' end extension 10 Nucleotide of core sequence A or the sequence obtained to 3 ' end extension, 10 Nucleotide of core sequence A;
Core sequence A:5 '-TCCTGCTGATGAAGCAGAACAA-3 ';
The sequence of described upstream primer F1130b is arbitrary sequence as described below: core sequence B; Or centered by core sequence B, hold to 5 ' and 3 ' sequence extending 1 ~ 10 Nucleotide respectively or simultaneously and obtain; The sequence be preferably core sequence B, obtaining to 5 ' end extension 10 Nucleotide of core sequence B or the sequence obtained to 3 ' end extension, 10 Nucleotide of core sequence B;
Core sequence B:5 '-ATATCGAACTCATGAAGCAGCATAA-3 ';
The sequence of described downstream primer R1264 is arbitrary sequence as described below: core sequence C; Or centered by core sequence C, the sequence of holding extension 1 ~ 10 Nucleotide to 5 ' and obtaining to 3 ' end extension, 1 ~ 2 Nucleotide respectively or simultaneously; The sequence be preferably core sequence C, obtaining to 5 ' end extension 10 Nucleotide of core sequence C or the sequence obtained to 3 ' end extension, 2 Nucleotide of core sequence C;
Core sequence C:5 '-CCATGCCGTGGGTTTCAATAT-3 ';
The sequence of described probe Pb1156A is core sequence D as follows; Or centered by core sequence D, the sequence of holding extension 1 ~ 2 Nucleotide to 5 ' and obtaining to 3 ' end extension, 1 ~ 10 Nucleotide respectively or simultaneously; The sequence be preferably core sequence D, obtaining to 5 ' end extension 2 Nucleotide of core sequence D or the sequence obtained to 3 ' end extension, 10 Nucleotide of core sequence D;
Core sequence D:5 '-R-AACGCCGTGCGCTGYTCGCA-Q-3 ', R are fluorescent reporter group, and Q is fluorescent quenching group;
Extension recited above all with the corresponding sequence of E. coli IacZ gene for template;
Described fluorescent reporter group is preferably FAM (6-Fluoresceincarboxylic acid), HEX (chlordene-6-methyl fluorescein) or TET (four chloro-6-Fluoresceincarboxylic acids);
Described fluorescent quenching group is preferably TAMRA (6-carboxyl tetramethylrhodamin) or BHQ1;
Described core sequence D is 5'-HEX-AACGCCGTGCGCTGYTCGCA-BHQ1-3'; Y is degeneracy base, is C or T;
The described test kit for detecting coliform, also comprises the enzyme for quantitative fluorescent PCR and reagent;
Described reagent comprises dNTP, damping fluid etc.;
The described test kit for detecting coliform can detection by quantitative coliform, is particularly suitable for applying in field of food.
The present invention has following advantage and effect relative to prior art:
(1) method of coliform is detected at present with molecular method, its primer and probe design mainly concentrate on 16S-23SrRNA, on 16S rRNA and lacZ (beta-galactosidase gene), due to 16S-23S rRNA, 16S rRNA copy number difference in four different generas (colon bacillus, citric acid bacillus, klebsiella and enterobacter) of coliform is very large, cause the molecular biology method of the detection coliform based on 16S-23S rRNA and 16S rRNA, cannot count intuitively coliform sum.Primer sequence provided by the present invention and probe sequence are based on designed by the lacZ of single copy, thus effectively can avoid aforementioned drawback.
(2) detection sensitivity of primer provided by the invention and probe can reach 56 copy/mL, illustrates that it has good sensitivity.
(3) primer provided by the invention and probe are for the detection sample standard deviation not containing coliform without amplified signal, illustrate that it has good specificity.
(4) because the present invention adopts Fluorescence PCR assay as detection method, whole reaction is all carried out in the reaction tubes closed, and avoids other nucleic acid detection methods and is easy to form Aerosol Pollution as PCR-electrophoresis etc. and causes false positive results; Owing to carrying out Real-Time Monitoring to PCR primer, greatly save monitoring time, save manpower and materials.
(5) test kit provided by the present invention can count the coliform in food or other samples fast and effectively, the man power and material greatly reducing used Detection Method for Coliform Group is consumed, simplifies the sampling observation of large batch of food or other samples in daily productive life.
Accompanying drawing explanation
Fig. 1 is the amplification curve diagram of different probe combination of primers.
Fig. 2 is the amplification curve diagram of partial target bacterial strain and non-targeted bacterial strain DNA sample; Wherein, 1-E. coli O157:H7CICC 21530,2-enterobacter cloacae Enterbacter cloacae CICC21539,3-enteroaerogen Enterbacteraerogenes CICC10293,4-citrobacter freundii Citrobacter freundii ATCC8090,5-Klebsiella pneumonia Klebsiella pneumonia CICC10781,6-acid-producing Klebsiella bacterium Klebsiella oxytoca CICC22912,7-salmonella paratyphi Salmonella paratyphi CMCC50093,8-negative control.
Fig. 3 is the Bland-Altman analytical results figure of MPN method and the inventive method acquired results.
Fig. 4 is the Bland-Altman analytical results figure of dull and stereotyped fast counting and the inventive method acquired results.
Fig. 5 is the Bland-Altman analytical results figure of MPN method and dull and stereotyped fast counting acquired results.
Wherein: MPN method represents GB/T4789.32-2002 method, dull and stereotyped fast counting represents SN/T 1896-2007 method, UCL and LCL is mean value ± 1.96SD reference line.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
1, primer and probe design: by comparing analysis to genomic 15 the lacZ gene orders of all known coliforms (colon bacillus, citric acid bacillus, klebsiella and enterobacteria) respectively, the section finding out high conservative has this 2 place of 1597-1643 and 1130-1169, selected without secondary structure and the section of high conservative by premier 3.0, design many groups primer and probe, about primer length is generally 20 bases, interior without complementary sequence with primer between primer.List of primers 1 is as follows:
Table 1 primer and probe list (being 5'-3')
For the primer shown in group 1 and probe sequence, its upstream primer and probe sequence and enterobacter cloacae (Enterobactercloacae) have two to three base pair difference, easily cause undetected.Therefore the secondary primer (F1130b) in the upstream of the main primer in the upstream (F1130a) that can comprise most of coliform lacZ gene target sequence and the lacZ gene target sequence that can include a small amount of enterobacter cloacae is selected can effectively to avoid undetected.The fragment that the primer of group 2 and group 3 can increase out is in theory analyzed by BLAST, display amplified fragments and other non-coliform bacterium, and as most Salmonellas, Shigellae and various gram-positive microorganisms etc., without intersecting.
2, step
(1) choose primer and probe, select the primer of group 3 and probe to test as follows;
(2) prepare template to be measured, adopt phenol-chloroform method (referring to " molecular cloning ") or test kit to extract the genomic dna of coliform in various source sample;
(3) foundation of reaction system: a, determine best primer concentration; B, determine magnesium ion concentration; C, determine Taq archaeal dna polymerase (Taq enzyme) consumption; D, determine dNTPs concentration; E, determine concentration and probe concentration;
Reaction conditions is as follows: 95 DEG C of 2min, 1 circulation; 95 DEG C of 5sec, 60 DEG C of 40sec, 40 circulations.
(4) select the sense channel of instrument: when carrying out Fluorescence PCR, in reply instrument, the collection of reaction tubes fluorescent signal is arranged, and the fluorescent reporter group that fluorescence detection channel and the probe of selection mark is consistent.Concrete method to set up is different because of instrument, should with reference to instrument working instructions.
(5) upper machine testing.
3, the foundation of reaction system and optimization: the foundation of reaction system and the target region template adopted in optimizing obtain in the following manner: intestinal bacteria (Escherichia coli) ATCC 9637 getting four Pseudomonas E Pseudomonas of coliform respectively, citrobacter freundii (Citrobacter freundii) ATCC 8090 of Citrobacter, cultivate 48 hours after acid-producing Klebsiella bacterium (Klebsiella oxytoca) CICC 21092 of Klebsiella and enterobacter cloacae (Enterbacter cloacae) the CICC21539 reference culture recovery of enterobacter, get nutrient solution 1ml and carry out 10 times of gradient dilutions, choose 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6totally 6 extent of dilution are as series of positive template, adopt phenol-chloroform method or test kit to extract genomic nucleic acids respectively, then carry out pcr amplification with above-mentioned primer and probe, and get wherein between Ct value 24-27 person and extent of dilution be 10
-4nutrient solution as template during later reaction system optimization.Wherein initial reaction system is as shown in table 2:
The PCR reaction system of table 2 initial design
| Component | Final concentration |
| 10 × PCR reaction buffer | 1× |
| Mg 2+Concentration | 2.0mmol/L |
| DNTPs (containing dUTP) | 0.15mmol/L |
| Taq enzyme | 2U |
| The main primer in upstream | 0.1μmol/L |
| The secondary primer in upstream | 0.1μmol/L |
| Downstream primer | 0.1μmol/L |
| Probe | 0.1μmol/L |
| Template | 2μl |
| Moisturizing extremely | 40μl |
The optimization of 3.1 primer concentrations: in reaction system, detects respectively by primer concentration after 0.1 μm of ol/L ~ 0.8 μm ol/L does multiple proportions serial dilution, by the com-parison and analysis of test-results, determines that best primer final concentration is 0.2 μm of ol/L.
The optimization of 3.2 magnesium ion concentrations: other condition is with under the prerequisite of table 1 in the final concentration of primer is 0.2 μm of ol/L and reaction system, by MgCl
2concentration increase progressively from 1mmol/L ~ 2.5mmol/L with 0.5mmol/L, be magnesium ion concentration in test kit reaction system through repeatedly repeating to test selected 2.5mmol/L.
The optimization of 3.3 Taq archaeal dna polymerase (Taq enzyme) consumptions: the final concentration of primer be 0.2 μm of ol/L, the final concentration of magnesium ion be 2.5mmol/L and reaction system in other condition with under the prerequisite of table 1, by comparing the optimization experiment result of Taq enzyme consumption (in unit Unit), selected 2U is as the consumption of Taq enzyme in test kit reaction system.
The optimization of 3.4 dNTPs concentration: the final concentration of primer be 0.2 μm of ol/L, the final concentration of magnesium ion be 2.5mmol/L and reaction system in other condition with under the prerequisite of table 1, by using the dNTPs of different concns to detect, after comprehensive assessment, select 0.2mmol/L as the usage quantity of dNTPs in test kit reaction system.
The optimization of 3.5 concentration and probe concentration: the final concentration of primer be 0.2 μm of ol/L, the final concentration of magnesium ion to be 2.5mmol/L be and in reaction system other condition with under the prerequisite of table 1, in reaction system, concentration and probe concentration is detected respectively after 0.05 μm of ol/L ~ 0.2 μm ol/L does multiple proportions serial dilution, by the com-parison and analysis of test-results, determine that best probe final concentration is 0.1 μm of ol/L.
Utilize above-mentioned primer and probe to carry out the foundation of reaction system, finally determine that the Fluorescence PCR system adopted is 40 μ l systems, required each component and respective concentration are in table 3.
PCR reaction system after table 3 optimization
| Component | Final concentration |
| 10 × PCR reaction buffer | 1× |
| Mg 2+Concentration | 2.5mmol/L |
| DNTPs (containing dUTP) | 0.2mmol/L |
| Taq enzyme | 2U |
| The main primer in upstream | 0.2μmol/L |
| The secondary primer in upstream | 0.2μmol/L |
| Downstream primer | 0.2μmol/L |
| Probe | 0.1μmol/L |
| Template | 2μl |
| Moisturizing extremely | 40μl |
Note: when Fluorescence PCR volume is different, each reagent should adjust in proportion.
4, the primer of group 2 and group 3 compares: the system using table 3, use intestinal bacteria (Escherichia coli) ATCC 9637 mentioned by step 3 and acid-producing Klebsiella bacterium (Klebsiella oxytoca) CICC 21092 to carry out Real-time PCR for template, condition is the same.As shown in Figure 1, result shows experimental result, has higher amplification efficiency when being R1266 when downstream primer is R1264 than downstream primer.Therefore obtain optimum primed probe and be combined as the primer shown in group 3 and probe sequence, as follows:
The main primers F 1130a:5'-TCCTGCTGATGAAGCAGAACAA-3' in upstream;
The secondary primers F 1130b:5'-ATATCGAACTCATGAAGCAGCATAA-3' in upstream;
Downstream primer R1264:5'-CCATGCCGTGGGTTTCAATAT-3';
Probe Pb1156A:5'-HEX-AACGCCGTGCGCTGYTCGCA-BHQ1-3'.
Embodiment 2
Choose primer pair F1130a, F1130b, R1264 and probe Pb1156A, by the nutrient solution phenol-chloroform method extracting genomic dna of coliform nutrient solution to be checked and other non-coliform bacterial strains totally 103 strain bacterium.Concrete steps are as follows:
(1) 103 strain bacterium as shown in table 4 are inoculated in respectively 37 DEG C of cultivations 48h, wherein vibrios Anaerobic culturel in Luria-Bertani substratum totally, the micro-aerobic cultivation of campylobacter jejuni, other bacterial strain shake-flask culture.Add in the centrifuge tube of 1.5ml by the enrichment liquid (about 1ml) to be checked of above-mentioned cultivation, centrifugal 5 minutes of 12000rpm, removes supernatant.
(2) add DNA cleavage liquid 700 μ l, fully mixing is resuspended, and water-bath boils 5 minutes.Wherein DNA cleavage liquid is 50mmol/L Tris-HCl, pH 8.0,100mmol/L NaCl, 25mmol/L EDTA (ethylenediamine tetraacetic acid (EDTA)), 2%SDS (sodium lauryl sulphate), 1.2%PVP (polyvinylpyrrolidone).
(3) isopyknic phenol/chloroform (V/V=1:1) solution is added, fully centrifugal after mixing, centrifugal 5 minutes of 13000rpm.
(4) supernatant liquor is moved in the centrifuge tube of another 1.5ml, add isopyknic chloroform, mixing, centrifugal 5 minutes of 13000rpm.
(5) supernatant liquor is moved in the centrifuge tube of another 1.5ml, add the Virahol of 0.6 times of volume, mixing of turning upside down, centrifugal 5 minutes of 13000rpm.
(6) use 70% alcohol flushing after abandoning supernatant, centrifugal 5 minutes of 13000rpm, careful suction abandons supernatant, and inversion is dried.
(7) add 50 μ l DNA lysates in centrifuge tube after the drying fully to mix, stand-by as DNA profiling.
In 40 μ l Fluorescence PCRs system (preparing by table 3), add the genomic dna 2 μ l of the different strains of above extraction, carry out fluorescent PCR detection according to the PCR reaction conditions in embodiment 1.
Experimental result is as shown in table 4 below.
The fluorescent quantitation qPCR result of table 4 different strains
A.+ is positive;-, negative
B.ATCC, American Type Culture Collecti; CMCC, Chinese medicine Culture Collection; CGMCC, China General Microbiological DSMZ; CICC, Research for Industrial Microbial Germ preservation administrative center; CCTCC, China typical culture collection center; SZCIQ, Shenzhen Entry-Exit Inspection and Quarantine Bureau; ADCPC, Animal diseases Control and prevention center, Chongqing; GZCDC, Guangzhou disease prevention and control center; SHCDC, Shanghai disease prevention and control center.
After testing, if in nutrient solution to be checked containing in coliform any one or more; display positive amplification curve, its detection sensitivity can reach 56 copy/ml; If not containing coliform in nutrient solution to be checked, without amplified signal, point out above-mentioned primer pair and probe to have good sensitivity and specificity.Wherein the amplification curve of partial target bacterial strain and non-targeted bacterial strain DNA sample as shown in Figure 2, amplification curve shown in figure is followed successively by with E. coli O157:H7 CICC 21530, Enterbacter cloacaeCICC21539, Enterbacter aerogenes CICC10293, Citrobacter freundii ATCC8090, Klebsiellapneumonia CICC10781, Klebsiella oxytoca CICC22912 and Salmonella paratyphi CMCC50093 is the amplification curve that template is carried out pcr amplification and obtained.Wherein in nutrient solution to be checked containing in coliform any one or more then show positive amplification curve, in nutrient solution to be checked only containing Salmonellas or be negative control time then without amplified signal, point out above-mentioned primer pair and probe to have good sensitivity and specificity.
Embodiment 3
Choose primer pair F1130a, F1130b, R1264 and probe Pb1156A, the coliform of pure culture is carried out gradient dilution, according to the method extracting DNA in embodiment 2, and carry out fluorescent PCR detection according to the PCR reaction conditions in embodiment 1.Its result and GB/T4789.32-2002 and SN/T 1896-2007 are compared, concrete steps are as follows:
E Pseudomonas E.coli O157:H7SZCIQ 13813 is inoculated in 37 DEG C of cultivation 16h in Luria-Bertani substratum, gets nutrient solution 1ml and carry out 10 times of gradient dilutions, choose bacterial content as shown in the table and be about 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2totally 10 diluent samples are as series of positive template with totally 8 extent of dilution within 10cfu/ml, and tested with qPCR method, GB/T4789.32-2002 and SN/T 1896-2007 respectively by each dilution bacterium liquid, result is as shown in table 5 below:
Table 5 qPCR method compares with traditional method
How a.TNTC (too numerous to count), can not count;
B.-, can not count lower than detectability;
C.SD, standard deviation
From upper table 5, traditional detection method only could accurately detect under suitable extent of dilution, and qPCR method then can meet 10 to 10
8scope in accurate quantitative analysis.In addition, the Bland-Altman result of 3 kinds of different methods gained carried out analyzes, and result as seen in figures 3-5.The method of Bland-Altman is observed the one of difference between two kinds of evaluations, and transverse axis is the average that each observed object evaluation obtains, and the longitudinal axis is the difference value between two kinds of evaluations, investigates the relation of mean and difference between evaluation person.Bland-Altman figure method mainly observes the distribution of difference between two kinds of measurements.On the longitudinal axis with the average of difference and theoretical value for average reference line, add the reference line of the fiducial interval of 95% of mean value ± 1.96SD and difference average in addition again, also referred to as the acceptable consistence boundary of 95%.As seen in figures 3-5, the difference of qPCR method and GB/T4789.32-2002 method (Fig. 3) acquired results is all within the fiducial interval of 95%, and therefore qPCR method and GB/T4789.32-2002 are 10
2to 10
3the order of magnitude within the scope of to the detected result of coliform, there is consistence; The difference of qPCR method and SN/T 1896-2007 method (Fig. 4) acquired results is all within the fiducial interval of 95%, and therefore qPCR method and SN/T 1896-2007 are 10
2to 10
3the order of magnitude within the scope of to the detected result of coliform, there is consistence; The difference of SN/T 1896-2007 and GB/T4789.32-2002 (Fig. 5) acquired results is all within the fiducial interval of 95%, and therefore SN/T 1896-2007 and GB/T4789.32-2002 is 10 to 10
3the order of magnitude within the scope of to the detected result of coliform, there is consistence.
Embodiment 4
Choose primer pair F1130a, F1130b, R1264 and probe Pb1156A, actual sample to be checked is prepared the even liquid of sample according to the method for GB/T4789.32-2002, the even liquid of sample of coliform may be contained according to the method extracting DNA in embodiment 2, and carry out fluorescent PCR detection according to the PCR reaction conditions in embodiment 1.Its result and GB/T4789.32-2002 and SN/T 1896-2007 are compared, concrete steps are as follows:
From 10 different supermarkets, stochastic buying 280 parts of cold fresh meat samples, detect coliform group count in sample according to traditional GB/T4789.32-2002 and SN/T 1896-2007 method and present method, statistics positive sample number respectively.According to the hygienic standard of the cold fresh meat of NY/T632-2002, object bacteria content is considered as the positive more than 100MPN/g or 100CFU/g, otherwise is then negative.
Result is as shown in table 6 below.
Coliform-positive rate in table 6 qPCR method and traditional technique in measuring actual sample
From upper table 6, the sample of qPCR, SN/T 1896-2007 and GB/T 4789.32-2002 method detects positive rate and is respectively 32.85%, 29.64% and 30.35%.Kappa consistency check is carried out to result, represents that as kappa>0.75 the consistence of two kinds of methods and resultses is good, show that as kappa<0.4 the consistence of two kinds of methods and resultses is poor.Kappa inspection shows that qPCR and GB/T4789.32-2002 method (kappa=0.893, P=0.00) result is consistent, qPCR and SN/T 1896-2007 method (kappa=0.909, P=0.00) result is consistent.Therefore qPCR method described in the invention can replace traditional Detection Method for Coliform Group in actual sample detects.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. for detecting a test kit for coliform, it is characterized in that: comprise primer sequence and probe sequence; Wherein: primer sequence is made up of upstream primer F1130a, upstream primer F1130b and downstream primer R1264; Probe sequence is probe Pb1156A;
The sequence of described upstream primer F1130a is arbitrary sequence as described below: core sequence A; Or centered by core sequence A, hold to 5 ' and 3 ' sequence extending 1 ~ 10 Nucleotide respectively or simultaneously and obtain;
Core sequence A:5 '-TCCTGCTGATGAAGCAGAACAA-3 ';
The sequence of described upstream primer F1130b is arbitrary sequence as described below: core sequence B; Or centered by core sequence B, hold to 5 ' and 3 ' sequence extending 1 ~ 10 Nucleotide respectively or simultaneously and obtain;
Core sequence B:5 '-ATATCGAACTCATGAAGCAGCATAA-3 ';
The sequence of described downstream primer R1264 is arbitrary sequence as described below: core sequence C; Or centered by core sequence C, the sequence of holding extension 1 ~ 10 Nucleotide to 5 ' and obtaining to 3 ' end extension, 1 ~ 2 Nucleotide respectively or simultaneously;
Core sequence C:5 '-CCATGCCGTGGGTTTCAATAT-3 ';
The sequence of described probe Pb1156A is core sequence D as follows; Or centered by core sequence D, the sequence of holding extension 1 ~ 2 Nucleotide to 5 ' and obtaining to 3 ' end extension, 1 ~ 10 Nucleotide respectively or simultaneously;
Core sequence D:5 '-R-AACGCCGTGCGCTGYTCGCA-Q-3 ', R are fluorescent reporter group, and Q is fluorescent quenching group.
2. the test kit for detecting coliform according to claim 1, is characterized in that: the sequence of described upstream primer F1130a is core sequence A, the sequence that obtains to 5 ' end extension, 10 Nucleotide of core sequence A or the sequence obtained to 3 ' end extension, 10 Nucleotide of core sequence A.
3. the test kit for detecting coliform according to claim 1, is characterized in that: the sequence of described upstream primer F1130b is core sequence B, the sequence that obtains to 5 ' end extension, 10 Nucleotide of core sequence B or the sequence obtained to 3 ' end extension, 10 Nucleotide of core sequence B.
4. the test kit for detecting coliform according to claim 1, is characterized in that: the sequence of described downstream primer R1264 is core sequence C, the sequence that obtains to 5 ' end extension, 10 Nucleotide of core sequence C or the sequence obtained to 3 ' end extension, 2 Nucleotide of core sequence C.
5. the test kit for detecting coliform according to claim 1, is characterized in that: the sequence of described probe Pb1156A is core sequence D, the sequence that obtains to 5 ' end extension, 2 Nucleotide of core sequence D or the sequence obtained to 3 ' end extension, 10 Nucleotide of core sequence D.
6. the test kit for detecting coliform according to claim 1, is characterized in that: described fluorescent reporter group is FAM, HEX or TET.
7. the test kit for detecting coliform according to claim 1, is characterized in that: described fluorescent quenching group is TAMRA or BHQ1.
8. the test kit for detecting coliform according to claim 1, is characterized in that: described core sequence D is 5'-HEX-AACGCCGTGCGCTGYTCGCA-BHQ1-3'.
9. the test kit for detecting coliform described in any one of claim 1 ~ 8, is characterized in that: also comprise the enzyme for quantitative fluorescent PCR and reagent.
10. the test kit for detecting coliform described in any one of claim 1 ~ 9 is applied in field of food.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410660228.3A CN104404144A (en) | 2014-11-18 | 2014-11-18 | Kit for detecting coli groups and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410660228.3A CN104404144A (en) | 2014-11-18 | 2014-11-18 | Kit for detecting coli groups and application |
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| Publication Number | Publication Date |
|---|---|
| CN104404144A true CN104404144A (en) | 2015-03-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410660228.3A Pending CN104404144A (en) | 2014-11-18 | 2014-11-18 | Kit for detecting coli groups and application |
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| CN (1) | CN104404144A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115265635A (en) * | 2022-09-29 | 2022-11-01 | 浙江中科凯泽科技有限公司 | Industrial machine vision detection management system based on data analysis |
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2014
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115265635A (en) * | 2022-09-29 | 2022-11-01 | 浙江中科凯泽科技有限公司 | Industrial machine vision detection management system based on data analysis |
| CN115265635B (en) * | 2022-09-29 | 2023-04-07 | 浙江中科凯泽科技有限公司 | Industrial machine vision detection management system based on data analysis |
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