CN101434992A - Fluorescent quantitative PCR detecting method for Listeria monocytogenes and Shigella - Google Patents
Fluorescent quantitative PCR detecting method for Listeria monocytogenes and Shigella Download PDFInfo
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- CN101434992A CN101434992A CNA2008101214271A CN200810121427A CN101434992A CN 101434992 A CN101434992 A CN 101434992A CN A2008101214271 A CNA2008101214271 A CN A2008101214271A CN 200810121427 A CN200810121427 A CN 200810121427A CN 101434992 A CN101434992 A CN 101434992A
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Abstract
The invention relates to a fluorescence quantitative PCR detection method of Listeria monocytogenes and Shigella, including the following steps: (1) the DNA extraction of a PCR template: (1.1) the DNA extraction of a sample to be detected: taking 1ml of the sample to be detected for conducting DNA extraction, 1ml of the sample to be detected-12000rpm, 2min, discarding supernatant-washing sediment for two times by PBS-200Mul TE suspension-adding 8Mul of lysozyme (10mg/ml) and 2Mul of RNaseA (10mg/ml), carrying out water bath with the temperature of 37 DEG C for 30min-adding lysate and proteinase K according to UNIQ-10DNA extraction kit, carrying out water bath with the temperature of 55 DEG C for 30min-extracting DNA according to the specification of the UNIQ-10DNA extraction kit; and (1.2) bacteria liquid DNA extraction; (2) construction of hly and ipaH plasmid standard product; and (3) fluorescence quantitative PCR amplification and analysis. The invention has the advantages that the invention uses double fluorescence quantitative PCR (TaqMan probe) for simultaneous quantitative detection of Listeria monocytogenes hly gene and Shigella ipaH gene, and provides a specific, rapid and accurate quantitative method for simultaneous quantitative detection of Listeria monocytogenes and Shigella for the food hygiene industry.
Description
Technical field
The present invention relates to the quantitative detecting method field, particularly relate to the fluorescent quantitative PCR detection method of a kind of Listeria monocytogenes and Shigellae.
Background technology
Listeria monocytogenes (Listeria monocytogenes) is important foodborne bacterial pathogens with Shigellae (Shigella), wherein the listeriosis (Listeriosis) that causes of Listeria monocytogenes can cause meningitis, septicemia and miscarriage, and pregnant woman, newborn infant, the elderly and immune deficiency patient are Susceptible population.Animal food is its main propagating source, and this bacterium still can grow at 4 ℃, and frozen prods and instant food are constituted potential threat.Shigellae is the serious pathogen enterobacteria of harm, ranks first in China infectious diarrhea pathogenic bacterium, and the Shigellae property dysentery (shigellosis) that it causes is in the world, especially one of important transmissible disease of developing country.
Listeria monocytogenes can cause that causing a disease is because it produces a plurality of virulence factors, is respectively the listeria bacteria hemolysin O, and aggressive albumen P60, internalization plain A, B and transcriptional regulatory subbase are because of prfA etc.Listeria bacteria hemolysin O by the hly genes encoding is the most important virulence factor of Listeria monocytogenes, most of PCR detection methods all with it as target gene, embodied very strong specificity.Shigellae is pathogenic, and aggressive plasmid antigen H gene ipaH is present on plasmid and the karyomit(e) simultaneously with a plurality of copy forms mainly by the virulence plasmid mediation, it is detected target gene as PCR have characteristics highly sensitive, high specificity.
Summary of the invention
The objective of the invention is to overcome the defective of above-mentioned technology, and provide the fluorescent quantitative PCR detection method of a kind of Listeria monocytogenes and Shigellae, and provide a kind of Auele Specific Primer and probe sequence that is used for fluorescence quantitative PCR detection Listeria monocytogenes and Shigellae.
The invention discloses Listeria monocytogenes in a kind of foodborne bacterial pathogens (Listeria monocytogenes) and Shigellae (Shigella spp) synchronous fluorescence quantitative PCR detecting method.
The objective of the invention is to be achieved through the following technical solutions.The fluorescent quantitative PCR detection method of this Listeria monocytogenes and Shigellae may further comprise the steps:
(1.1), pcr template DNA extraction:
(1.1.1), testing sample DNA extraction: get the 1ml testing sample and carry out DNA extraction, get wherein 4 dilution 0.1ml bacterium liquid coating BHI flat boards,, carry out colony count in 37 ℃ of cultivation 24h; 1ml testing sample-12000rpm, 2min, abandon supernatant-precipitation with PBS wash twice-200 μ l TE resuspended-add 8 μ l N,O-Diacetylmuramidases (10mg/ml) and 2 μ l RNaseA (10mg/ml), 37 ℃ of water-bath 30min-extract test kit according to UNIQ-10DNA and add lysate and Proteinase K, extract DNA with reference to UNIQ-10 DNA extraction test kit specification sheets behind 55 ℃ of water-bath 30min-;
(1.1.2), bacterium liquid DNA extraction: get the bacterium liquid of incubated overnight, adopt UNIQ-10DNA to extract test kit and extract;
(1.2), the structure of hly and ipaH plasmid standard:
(1.3), fluorescent quantitative PCR and analysis: will calculate the Listeria monocytogenes and the Shigellae plasmid DNA of original copy number, and press 10 times of gradient dilutions, peek magnitude 10
5Copies/ μ l to 10
1Copies/ μ l adds 25 μ l reaction systems as template, at IQ
TMThe enterprising performing PCR amplification of 5 quantitative real time PCR Instruments obtains typical curve; Pcr amplification reaction system (25 μ l) wherein: each 1 μ l of template DNA; 2 * Premix ExTaq
TM12.5 μ l; Each 1 μ l of 10 μ mol/L primers; Each 0.5 μ l of 10 μ mol/L probes; Add sterilization ultrapure water to 25 μ l, reaction conditions: 95 ℃ of 3min; 95 ℃ of 15s; 58.5 ℃ 1min, 45 circulations.
The fluorescent quantitative PCR detection method of described Listeria monocytogenes and Shigellae, the structure of described hly and ipaH plasmid standard, concrete steps are:
(1.2.1), respectively by hly and ipaH primer amplification Listeria monocytogenes hly and Shigellae ipaH gene, amplification system (25 μ l): template DNA 1 μ l; 10 * Taq damping fluid, 2 μ l; Mg
2+1.5mmol/L; DNTPs 0.25mmol/L; Upstream and downstream primer 500nmol/L; Taq archaeal dna polymerase 2U; Add sterilization ultrapure water to 25 μ l, reaction conditions: pre-94 ℃ of 4min of sex change; 94 ℃ of 30s of sex change; 59 ℃ of 30s anneal; Extend 72 ℃ of 20s; Totally 35 circulations, last 72 ℃ of 10min;
(1.2.2), amplified production carries out 1.8% agarose gel electrophoresis earlier, again behind PCR product purification test kit purifying, it is connected into the pGEM-T carrier, with gained recombinant plasmid transformed DH5 α bacterium (empty plasmid intestinal bacteria, competent cell), chooses the mono-clonal bacterium colony and cultivate, extract plasmid with plasmid extraction kit, carry out PCR detection, electrophoresis evaluation, dna sequencing;
(1.2.3), recombinant plasmid carries out the linearizing of EcoRI single endonuclease digestion, reclaims the linear recombinant plasmid that obtains and measures concentration and purity through ultraviolet spectrophotometer by cutting glue ,-20 ℃ of preservations are standby.
The fluorescent quantitative PCR detection method of described Listeria monocytogenes and Shigellae, the sequence of the primer 1 (hly1) of described hly gene is 5 '-ACAACTTCAAAAGCTTATACAGATGGA-3 ', product size 188bp; The sequence of primer 2 (hly2) is 5 '-GGCAAATAGATGGACGATGTGAAAT-3 '; The sequence of probe 1 (probe1) is 5 '-TCGATCACTCTGGAGGATACGTTGCTC-3 '.
The fluorescent quantitative PCR detection method of described Listeria monocytogenes and Shigellae, the sequence of the primer 2 of described ipaH gene (ipaH1) is 5 '-TGCCTCTGCGGAGCTTCG-3 ', product size 129bp; The sequence of primer 2 (ipaH2) is 5 '-CCTTCTGATGCCTGATGGACCA-3 '; The sequence of probe 2 (probe2) is 5 '-TTCGCTGTTGCTGCTGATGCCACTGA-3 '.
Beneficial effect of the present invention: the present invention uses double fluorescent quantitative PCR (TaqMan probe) detection by quantitative Listeria monocytogenes hly gene and Shigellae ipaH gene synchronously, and the method for a kind of special, quick, quantitatively synchronous accurately detection by quantitative Listeria monocytogenes and Shigellae is provided for the food sanitation industry.
Description of drawings
Fig. 1 is pGEM-T Vector of the present invention (T carrier) collection of illustrative plates synoptic diagram;
Fig. 2 is amplification curve and the canonical plotting that double fluorescent quantitative PCR of the present invention detects the Listeria monocytogenes recombinant plasmid dna;
Fig. 3 is amplification curve and the canonical plotting that double fluorescent quantitative PCR of the present invention detects the Shigellae recombinant plasmid dna;
Fig. 4 is the amplification curve and the canonical plotting of double fluorescent quantitative PCR synchronous detection Listeria monocytogenes of the present invention and Shigellae recombinant plasmid dna;
Fig. 5 is the amplification curve diagram that double fluorescent quantitative PCR of the present invention detects artificial contamination's degreasing sterilization breast;
Embodiment
For making the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with drawings and the specific embodiments:
The fluorescent quantitative PCR detection method of this Listeria monocytogenes and Shigellae may further comprise the steps:
(1.1), pcr template DNA extraction:
(1.1.1), testing sample DNA extraction: get the 1ml testing sample and carry out DNA extraction, get wherein 4 dilution 0.1ml bacterium liquid coating BHI flat boards,, carry out colony count in 37 ℃ of cultivation 24h; 1ml testing sample-12000rpm, 2min, abandon supernatant-precipitation with PBS wash twice-200 μ lTE resuspended-add 8 μ l N,O-Diacetylmuramidases (10mg/ml) and 2 μ l RNaseA (10mg/ml), 37 ℃ of water-bath 30min-extract test kit according to UNIQ-10DNA and add lysate and Proteinase K, extract DNA with reference to UNIQ-10 DNA extraction test kit specification sheets behind 55 ℃ of water-bath 30min-;
(1.1.2), bacterium liquid DNA extraction: get the bacterium liquid of incubated overnight, adopt UNIQ-10DNA to extract test kit and extract;
(1.2), the structure of hly and ipaH plasmid standard:
(1.2.1), respectively by hly and ipaH primer amplification Listeria monocytogenes hly and Shigellae ipaH gene, amplification system (25 μ l): template DNA 1 μ l; 10 * Taq damping fluid, 2 μ l; Mg
2+1.5mmol/L; DNTPs 0.25mmol/L; Upstream and downstream primer 500nmol/L; Taq archaeal dna polymerase 2U; Add sterilization ultrapure water to 25 μ l, reaction conditions: pre-94 ℃ of 4min of sex change; 94 ℃ of 30s of sex change; 59 ℃ of 30s anneal; Extend 72 ℃ of 20s; Totally 35 circulations, last 72 ℃ of 10min;
(1.2.2), amplified production carries out 1.8% agarose gel electrophoresis earlier, again behind PCR product purification test kit purifying, it is connected into pGEM-T carrier (Fig. 1, available from Promega company), with gained recombinant plasmid transformed DH5 α bacterium (empty plasmid intestinal bacteria, competent cell), choosing the mono-clonal bacterium colony cultivates, extract plasmid with plasmid extraction kit, carry out PCR detection, electrophoresis evaluation, dna sequencing; Dna sequencing result and GenBank sequence alignment show, the DQ844159 that hly gene order that recombinant plasmid is contained and Genbank announce carries out the nucleotide sequence comparison, homology is 100%, and the CP000035 homology that ipaH gene order and Genbank announce is 99%.By DNAMAN software analysis EcoRI restriction enzyme site, be presented in hly and the ipaH amplified fragments all not this site, can carry out linearization for enzyme restriction.
(1.2.3), recombinant plasmid carries out the linearizing of EcoRI single endonuclease digestion, reclaims the linear recombinant plasmid that obtains and measures concentration and purity through ultraviolet spectrophotometer by cutting glue ,-20 ℃ of preservations are standby.
(1.3), fluorescent quantitative PCR and analysis: will calculate the Listeria monocytogenes and the Shigellae plasmid DNA of original copy number, and press 10 times of gradient dilutions, peek magnitude 10
5Copies/ μ l to 10
1Copies/ μ l adds 25 μ l reaction systems as template, at IQ
TMThe enterprising performing PCR amplification of 5 quantitative real time PCR Instruments obtains typical curve; Pcr amplification reaction system (25 μ l) wherein: each 1 μ l of template DNA; 2 * Premix ExTaq
TM12.5 μ l; Each 1 μ l of 10 μ mol/L primers; Each 0.5 μ l of 10 μ mol/L probes; Add sterilization ultrapure water to 25 μ l, reaction conditions: 95 ℃ of 3min; 95 ℃ of 15s; 58.5 ℃ 1min, 45 circulations.
With 10 times of gradient dilutions to final concentration 10
5Copies/ μ l to 10
1The linear recombinant plasmid dna of the Listeria monocytogenes of copies/ μ l and Shigellae carries out the double fluorescent quantitative PCR amplification.Reaction finishes back IQ
TM5 quantitative real time PCR Instruments carry out interpretation of result, and analysis software carries out data analysis.Survey corresponding relation between Ct value (y) and the contained plasmid DNA copy number concentration (x) according to each reaction tubes, obtain the typical curve that double fluorescent quantitative PCR detects Listeria monocytogenes: y=-3.482log (x)+41.275 (R
2=0.999) sees Fig. 1; Double fluorescent quantitative PCR detects the typical curve of Shigellae: y=-3.577log (x)+41.494 (R
2=0.999) sees Fig. 2; The typical curve that double fluorescent quantitative PCR detects synchronous detection Listeria monocytogenes and Shigellae is respectively: y=-3.780log (x)+43.320 (R
2=0.998); Y=-3.5741og (x)+42.068 (R
2=0.999) sees Fig. 3.It is suitable that the amplification efficiency of double fluorescent quantitative PCR synchronous detection Listeria monocytogenes and Shigellae, detection lower bound and quantitative linearity scope and double fluorescent quantitative PCR detect wherein a kind of bacterium separately, and 10
1The Ct of copies all＜40 has only the amplification efficiency of Listeria monocytogenes hly gene to descend to some extent, but detection lower bound and quantitative linearity scope are not affected.
Fig. 2 double fluorescent quantitative PCR detects the amplification curve and the typical curve 1-5:10 of Listeria monocytogenes recombinant plasmid dna
510
410
310
210
1Copy number FAM Listeria monocytogenes is a fluorescent mark.
Fig. 3 double fluorescent quantitative PCR detects the amplification curve and the typical curve 1-5:10 of Shigellae recombinant plasmid dna
510
410
310
210
1Copy number HEX is that Shigellae is a fluorescent mark.
The amplification curve of Fig. 4 double fluorescent quantitative PCR synchronous detection Listeria monocytogenes and Shigellae recombinant plasmid dna and typical curve 1-5:10
510
410
310
210
1Copy number FAM Listeria monocytogenes is that Shigellae is a fluorescent mark for fluorescent mark HEX.
Artificial contamination's degreasing sterilization breast double fluorescent quantitative PCR detects:
With the bacterium amount is 10
6-10
1The artificial degreasing sterilization breast of CFU/ml extracts DNA and carries out the double fluorescent quantitative PCR detection, and the result shows that the detection lower bound of double fluorescent quantitative PCR detection Listeria monocytogenes and Shigellae is 10
2CFU/ml, and 10
1CFU/ml and negative control do not see that fluorescent signal produces, and see Fig. 5, and double fluorescent quantitative PCR detects the amplification curve 1-5:10 of artificial contamination's degreasing sterilization breast
610
510
410
310
2CFU/ml; 6:10
1CFU/ml; 7: negative control, PCR base line subtracted Curve Fit RFU, Cycle cycle number primer and probe
Listeria monocytogenes hly gene (listeria monocytogenes hemolysin O gene) sequence (sequence number: according to the Genbank announcement DQ844159) with Shigellae ipaH gene (Shigellae aggressive plasmid antigen H gene) sequence (sequence number: CP000035), utilize Beacon Designer2.0 design primer and probe, it is synthetic to give birth to the worker by Shanghai.Hly probe 5 ' end flag F AM, 3 ' end mark TAMRA; IpaH probe 5 ' end mark HEX, 3 ' end mark TAMRA, the primer and the probe sequence of design see Table 1.
Table 1 primer, probe sequence and amplified production
Making method is as follows in the testing sample in experiment:
1, microbial culture
-70 ℃ of Listeria monocytogenes and Shigellaes that are stored in 15% glycerine are inoculated into recovery cultivation 16h in the nutrient broth.The Listeria monocytogenes streak inoculation is in PALCAM agar, and the Shigellae streak inoculation is in SS agar, and 37 ℃, 48h.Respectively picking Listeria monocytogenes, the single colony inoculation of Shigellae be in LB1 and LB, and 37 ℃, 200r/min shaking culture 12-16h are collected thalline and are used for DNA extraction.
PALCAM agar, SS agar, LB1 and LB are the special culture media title.Wherein, the PALCAM nutrient agar is used for singly increasing the selective separation (FDABAM, ISO) of Lee Salmonella; SS agar is used for Salmonellas, and the selective separation of Shigellae is cultivated; LB1 is used for the Li Shi bacterium two-step and increases bacterium; LB meat soup is used for microbial culture.
2, artificial contamination's sample
With 0.9% physiological saline Listeria monocytogenes and 10 times of gradient dilutions of Shigellae bacterium liquid with incubated overnight, with each dilution bacterium liquid artificial contamination's food (available from the degreasing sterilization breast in local supermarket), get 1ml degreasing sterilization breast and carry out DNA extraction, get wherein 4 dilution 0.1ml bacterium liquid coating BHI flat boards, in 37 ℃ of cultivation 24h, carry out colony count.
The foregoing description is used for the present invention that explains, rather than limits the invention, and in the protection domain of spirit of the present invention and claim, any modification and change to the present invention makes all fall into protection scope of the present invention.
Claims (4)
1, the fluorescent quantitative PCR detection method of a kind of Listeria monocytogenes and Shigellae is characterized in that: may further comprise the steps:
(1.1), pcr template DNA extraction:
(1.1.1), testing sample DNA extraction: get the 1ml testing sample and carry out DNA extraction, get wherein 4 dilution 0.1ml bacterium liquid coating BHI flat boards,, carry out colony count in 37 ℃ of cultivation 24h; 1ml testing sample-12000rpm, 2min, abandon supernatant-precipitation with PBS wash twice-200 μ l TE resuspended-add 8 μ l N,O-Diacetylmuramidases (10mg/ml) and 2 μ l RNaseA (10mg/ml), 37 ℃ of water-bath 30min-add lysate and Proteinase K according to UNIQ-10 DNA extraction test kit, extract DNA with reference to UNIQ-10 DNA extraction test kit specification sheets behind 55 ℃ of water-bath 30min-;
(1.1.2), bacterium liquid DNA extraction: get the bacterium liquid of incubated overnight, adopt UNIQ-10DNA to extract test kit and extract;
(1.2), the structure of hly and ipaH plasmid standard:
(1.3), fluorescent quantitative PCR and analysis: will calculate the Listeria monocytogenes and the Shigellae plasmid DNA of original copy number, and press 10 times of gradient dilutions, peek magnitude 10
5Copies/ μ l to 10
1Copies/ μ l adds 25 μ l reaction systems as template, at IQ
TMThe enterprising performing PCR amplification of 5 quantitative real time PCR Instruments obtains typical curve; Pcr amplification reaction system (25 μ l) wherein: each 1 μ l of template DNA; 2 * Premix ExTaq
TM12.5 μ l; Each 1 μ l of 10 μ mol/L primers; Each 0.5 μ l of 10 μ mol/L probes; Add sterilization ultrapure water to 25 μ l, reaction conditions: 95 ℃ of 3min; 95 ℃ of 15s; 58.5 ℃ 1min, 45 circulations.
2, the fluorescent quantitative PCR detection method of Listeria monocytogenes according to claim 1 and Shigellae is characterized in that: the structure of described hly and ipaH plasmid standard, and concrete steps are:
(1.2.1), respectively by hly and ipaH primer amplification Listeria monocytogenes hly and Shigellae ipaH gene, amplification system (25 μ l): template DNA 1 μ l; 10 * Taq damping fluid, 2 μ l; Mg
2+1.5mmol/L; DNTPs 0.25mmol/L; Upstream and downstream primer 500nmol/L; Taq archaeal dna polymerase 2U; Add sterilization ultrapure water to 25 μ l, reaction conditions: pre-94 ℃ of 4min of sex change; 94 ℃ of 30s of sex change; 59 ℃ of 30s anneal; Extend 72 ℃ of 20s; Totally 35 circulations, last 72 ℃ of 10min;
(1.2.2), amplified production carries out 1.8% agarose gel electrophoresis earlier, again behind PCR product purification test kit purifying, it is connected into the pGEM-T carrier, with gained recombinant plasmid transformed DH5 α bacterium (empty plasmid intestinal bacteria, competent cell), chooses the mono-clonal bacterium colony and cultivate, extract plasmid with plasmid extraction kit, carry out PCR detection, electrophoresis evaluation, dna sequencing;
(1.2.3), recombinant plasmid carries out the linearizing of EcoRI single endonuclease digestion, reclaims the linear recombinant plasmid that obtains and measures concentration and purity through ultraviolet spectrophotometer by cutting glue ,-20 ℃ of preservations are standby.
3, the fluorescent quantitative PCR detection method of Listeria monocytogenes according to claim 1 and Shigellae, it is characterized in that: the sequence of the primer 1 (hly1) of described hly gene is 5 '-ACAACTTCAAAAGCTTATACAGATGGA-3 ', product size 188bp; The sequence of primer 2 (hly2) is 5 '-GGCAAATAGATGGACGATGTGAAAT-3 '; The sequence of probe 1 (probel) is 5 '-TCGATCACTCTGGAGGATACGTTGCTC-3 '.
4, the fluorescent quantitative PCR detection method of Listeria monocytogenes according to claim 1 and Shigellae is characterized in that: the sequence of the primer 2 of described ipaH gene (ipaH1) is 5 '-TGCCTCTGCGGAGCTTCG-3 ', product size 129bp; The sequence of primer 2 (ipaH2) is 5 '-CCTTCTGATGCCTGATGGACCA-3 '; The sequence of probe 2 (probe2) is 5 '-TTCGCTGTTGCTGCTGATGCCACTGA-3 '.
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| CN111621581A (en) * | 2020-06-28 | 2020-09-04 | 河南大学 | Standard plasmid freeze-dried powder for detecting listeria monocytogenes |
| CN111719007A (en) * | 2020-08-06 | 2020-09-29 | 南方医科大学 | Listeria monocytogenes nucleic acid detection plasmid DNA reference sample, preparation method and application thereof |
| CN112852986A (en) * | 2021-03-18 | 2021-05-28 | 石家庄市畜产品和兽药饲料质量检测中心(石家庄市畜产品质量研究所) | Primer, probe and kit for detecting listeria monocytogenes by RAA fluorescence method |
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