CN107964566A - Salmonella in Food PCR detection primers, probe and detection method - Google Patents
Salmonella in Food PCR detection primers, probe and detection method Download PDFInfo
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- CN107964566A CN107964566A CN201711390721.8A CN201711390721A CN107964566A CN 107964566 A CN107964566 A CN 107964566A CN 201711390721 A CN201711390721 A CN 201711390721A CN 107964566 A CN107964566 A CN 107964566A
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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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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The present invention designs primer and probe according to the gene order of salmonella typhimurium gene invA genes, establishes the method based on Taqman probe for real-time fluorescence PCR detection salmonella typhimurium.Real time fluorescent PCR detection method is carried out to sample with the detection primer of the present invention and probe the method according to the invention, detection speed is fast, specificity is good, disturbed from false positive and cross contamination etc., reliable results, and high sensitivity, high specificity, it is adapted to Food Inspection, Center for Disease Control, quality supervised department to use.
Description
Technical field
The invention belongs to technical field of food detection, and in particular to salmonella typhimurium PCR detection primers, spy in food
Pin and detection method.
Background technology
In the prior art, GB 4789.4-2016《National food safety standard Food microbe testing salmonella is examined》
The idiographic flow of middle detection salmonella is shown in Fig. 1, and the method detection salmonella expends, it is necessary to time length (about 5 days) and examines consumption
Material is more (6 kinds of culture mediums, a set of biochemical identification kit and Salmeterol fluticasone propionate serum), spends human and material resources big.
The content of the invention
It is an object of the invention to provide salmonella typhimurium PCR detection method in quickly and easily food.
To achieve the above object, salmonella typhimurium PCR detection primers, probe in food,
For the invA genes of salmonella typhimurium gene,
The primer is：Sense primer：GAG TAT TGA TGC CGA TTT GAA GG
Anti-sense primer：TTC ATC GCA CCG TCA AAG GAA C
The probe is：/56-FAM/CG AAG CGT A/ZEN/C TGG AAA GGG AAA GC/3IABkFQ/.
The probe is connected with fluorophor FAM at 5 ＇-end, be connected with respectively at middle and 3 ＇-end quenching fluorescence group ZEN and
IABkFQ, such design can reduce background signal, improve inspection side sensitivity.
Using the method for salmonella typhimurium in above-mentioned primer, probe in detecting food, comprise the following steps：
(1) Zengjing Granule is carried out to sample, extracts the genomic DNA of thalline in enrichment liquid as template；
(2) above-mentioned detection primer and probe, reaction solution and hot resistant DNA polymerase with real-time fluorescent PCR amplification are used
It is mixed to form amplification reaction system；
(3) amplification reaction system is subjected to real-time fluorescence PCR reaction on fluorescent PCR instrument, after reaction, according to probe
The fluorescence signal of the fluorescent reporter group record of fluorescent marker, reads and records the PCR amplification cycle-index of each detection Yang Ping
(Ct)；
(4) according to the Ct values of various samples, according to the criterion of foundation, whether mouse typhus sramana is contained in judgement sample
Salmonella.
Wherein, fluorescent PCR amplification reaction system includes following components：
Wherein, PCR response procedures follow the steps below：
(1) 95 DEG C of pre-degeneration 5min；
(2) 95 DEG C are denatured 10sec, 55 DEG C of annealing 30sec, circular response totally 35 circulations.
The advantages and positive effects of the present invention are：
The present invention designs primer and probe according to the gene order of salmonella typhimurium invA genes, establishes and is based on
The method that Taqman probe for real-time fluorescence PCR detects salmonella typhimurium.With the detection primer of the present invention and probe according to this
The method of invention carries out sample real time fluorescent PCR detection method, and detection speed is fast (PCR detection process 1h or so completions), specificity
It is good, disturbed from false positive and cross contamination etc., reliable results, and high sensitivity, high specificity, it is adapted to Food Inspection, disease
Control centre, quality supervised department use.
Brief description of the drawings
Salmonella typhimurium check problem in Fig. 1 GB 4789.10-2016；
Fig. 2 is Real-TimePCR cyclic amplifications figure in embodiment；
Fig. 3 is real-time fluorescence quantitative PCR product sequencer map in embodiment, wherein, ordinate is signal strength, and abscissa is
Read length.
Embodiment
In order to understand the present invention, the present invention is further illustrated with embodiment below, but do not limit the present invention.
Embodiment salmonella typhimurium fluorescent PCR test experience
First, raw material introduction of apparatus
1.1 experiment bacteriums：
Salmonella typhimurium.
1.2 experiment reagent：
AceQ qPCR Probe Master Mix are purchased from Vazyme companies；Bacterial genomes DNA extraction kit is purchased
From Solarbio companies；Xylose lysine deoxidation cholate (XLD) agar, buffered peptone water (BPW) are purchased from Beijing overpass technology
Limited company.
1.3 laboratory apparatus：
Bio-Rad iQTM5 real-time fluorescence quantitative PCR instrument, purchased from BIO-RAD companies of the U.S.；SPX-100 biochemical cultivation cases are purchased
From Science and Technology Ltd. of Zhong Yiguo sections；SPH-200B constant temperature culture oscillators are purchased from Shanghai Shiping Experiment Equipment Co., Ltd.；
YXQ-LS-75G vertical pressure steam sterilizers are purchased from Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd..
2nd, experimental procedure：
The culture of 2.1 salmonella typhimuriums
2.1.1 salmonella typhimurium recovers
Take -80 to freeze bacterial strain, melt, dipped using 10 μ L oeses in bacterium solution line culture and XLD solid mediums, 37
DEG C constant temperature incubation 24-48h.
2.1.2 salmonella typhimurium amplification cultivation
After line culture 24h, picking single bacterium colony is seeded in 9ml BPW, 37 DEG C of shaken cultivations, until liquid is muddy.Use
Fresh 2-3 rear extraction genome of medium culture carries out fluorescent PCR experiment.
2.2 real-time fluorescence quantitative PCR
2.1 DNA of bacteria extract
The suspension of the salmonella typhimurium of 1ml amplification cultivations is taken, according to bacterial genomes DNA extraction kit specification
The step of extract salmonella typhimurium DNA.
2.2.2 Real-Time PCR
2.2.2.1 the design and synthesis of primer and probe
Compared repeatedly with the BLAST functions on NCBI and DNAman softwares according to the GenBank invA genes announced, then profit
With Oligo5.0 software Design primers and TaqMan probe, primer and probe sequence is shown in Table 1.
1 salmonella typhimurium upstream of table, anti-sense primer, probe
2.2.2.2 25 μ L PCR reaction systems are established
2 Real-Time PCR reaction systems of table
2.2.2.3, Real-Time PCR reaction conditions are set
3 Real-Time PCR reaction conditions of table
3rd, experimental result：
3.1 Real-TimePCR cyclic amplification results
By 35 Real-TimePCR cyclic amplifications, the result is shown in Fig. 2 and table 4.Add salmonella typhimurium DNA moulds
There is fluorescence response at 14.66 in plate group, and negative control group does not have fluorescence response, illustrates the primer and probe pair that the present invention designs
Salmonella typhimurium gene has good specificity.
4 Real-TimePCR cyclic amplification results of table
The specificity of the analysing amplified products of 3.2 BLAST
Fluorescent PCR amplified production is subjected to gene sequencing, the result is shown in Fig. 3.
As seen from Figure 3, sequencing result peak shape is relatively sharp, does not cover peak, and sequencing result shows that amplification gene is
InvA genes, BLAST analysis results show no homology or sequence identity biological gene information, further demonstrate fluorescence
The reliability and specificity of PCR experiment result.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on, should all be included in the protection scope of the present invention god.
Claims (4)
1. salmonella typhimurium PCR detection primers, probe in food, it is characterised in that
For the invA genes of salmonella typhimurium gene
The primer is：Sense primer：GAG TAT TGA TGC CGA TTT GAA GG
Anti-sense primer：TTC ATC GCA CCG TCA AAG GAA C
The probe is：/56-FAM/CG AAG CGT A/ZEN/C TGG AAA GGG AAA GC/3IABkFQ/.
A kind of 2. method for detecting salmonella typhimurium in food, it is characterised in that comprise the following steps：
(1) Zengjing Granule is carried out to sample, extracts the genomic DNA of thalline in enrichment liquid as template；
(2) detection primer and probe described in usage right requirement 1, reaction solution and heat-resistant dna with real-time fluorescent PCR amplification
Polymerase is mixed to form amplification reaction system；
(3) amplification reaction system is subjected to real-time fluorescence PCR reaction on fluorescent PCR instrument, after reaction, according to fluorescence probe
The fluorescence signal of the fluorescent reporter group record of mark, reads and records the PCR amplification cycle-index (Ct) of each detection Yang Ping；
(4) according to the Ct values of various samples, according to the criterion of foundation, whether Salmonella typhimurium is contained in judgement sample
Bacterium.
3. according to the method for salmonella typhimurium in a kind of detection food in claim 2, it is characterised in that fluorescent PCR
Amplification reaction system includes following components：
0.5 μ L of sense primer；
0.5 μ L of anti-sense primer；
0.3 μ L of probe；
2 μ L of template DNA；
DNA master mix 12.5μL；
9.2 μ L of sterile purified water.
4. according to the method for salmonella typhimurium in a kind of detection food in claim 2, it is characterised in that PCR reacts
Program follows the steps below：
(1) 95 DEG C of pre-degeneration 5min；
(2) 95 DEG C are denatured 10sec, 55 DEG C of annealing 30sec, circular response totally 35 circulations.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391451A (en) * | 2021-01-21 | 2021-02-23 | 江西省药品检验检测研究院 | Method for pretreating Chinese medicinal preparation |
Citations (3)
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|---|---|---|---|---|
| CN101760531A (en) * | 2008-11-21 | 2010-06-30 | 叶军 | Method for detecting vibrio parahaemolyticus and salmonella by TaqMan-PCR (Polymerase Chain Reaction) |
| CN102220427A (en) * | 2011-05-10 | 2011-10-19 | 浙江省质量技术监督检测研究院 | Multiple PCR (Polymerase Chain Reaction) detection method for four food-borne pathogens, detection primer set and kit |
| CN102618635A (en) * | 2012-02-21 | 2012-08-01 | 四川农业大学 | FR-PCR detection method for specificity of salmonella typhimurium |
-
2017
- 2017-12-21 CN CN201711390721.8A patent/CN107964566A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101760531A (en) * | 2008-11-21 | 2010-06-30 | 叶军 | Method for detecting vibrio parahaemolyticus and salmonella by TaqMan-PCR (Polymerase Chain Reaction) |
| CN102220427A (en) * | 2011-05-10 | 2011-10-19 | 浙江省质量技术监督检测研究院 | Multiple PCR (Polymerase Chain Reaction) detection method for four food-borne pathogens, detection primer set and kit |
| CN102618635A (en) * | 2012-02-21 | 2012-08-01 | 四川农业大学 | FR-PCR detection method for specificity of salmonella typhimurium |
Non-Patent Citations (4)
| Title |
|---|
| JEREMY PRITCHARD: "Better PCR probes: A second quencher lowers background,increasing signal detection", 《BETTER PCR PROBES: A SECOND QUENCHER LOWERS BACKGROUND,INCREASING SIGNAL DETECTION》 * |
| NARJOL GONZA´LEZ-ESCALONA等: "Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| PROMEGA公司: "Salmonella enterica DNA Purification from Food", 《SALMONELLA ENTERICA DNA PURIFICATION FROM FOOD》 * |
| 荣策等: "实时荧光PCR法检测鼠伤寒沙门氏菌", 《食品安全质量检测学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112391451A (en) * | 2021-01-21 | 2021-02-23 | 江西省药品检验检测研究院 | Method for pretreating Chinese medicinal preparation |
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Application publication date: 20180427 |