CN102304560B - Fluorescence quantitative polymerase chain reaction (PCR) method for detecting lactobacillus fermentum quickly - Google Patents
Fluorescence quantitative polymerase chain reaction (PCR) method for detecting lactobacillus fermentum quickly Download PDFInfo
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Abstract
The invention relates to a fluorescence quantitative polymerase chain reaction (PCR) method for detecting lactobacillus fermentum quickly. The fluorescence quantitative polymerase chain reaction (PCR) method comprises the following steps of: extracting deoxyribonucleic acid (DNA) of a genome of a suspected bacterial colony on a separation agar plate of a sample to be detected, and diluting for later use; designing specific primers by utilizing a conservative DNA sequence of a genome of the lactobacillus fermentum; performing fluorescence quantitative PCR amplification and detection on the DNA of the genome of the suspected bacterial colony on the separated agar plate of the sample to be detected by using the primers; adding 25 micro liters of double distilled H2O (dd H2O); setting process parameters of a fluorescence quantitative PCR instrument, namely the temperature is 37 DEG C and the time is 2 minutes; pre-modifying at the temperature of 95 DEG C for 30 seconds, modifying at the temperature of 95 DEG C for 5 seconds, annealing at the temperature of 60 DEG C for 40 seconds, collecting carboxy fluorescein (FAM) fluorescence, and circulating for 40 times; and storing at the temperature of 4 DEG C, wherein in sample loading parameters, the total volume of each fluorescence quantitative PCR reaction is 25 micro liters, and each fluorescence quantitative PCR reaction uses 2 micro liters of DNA of the extracted genome, 2.5 micro liters of 10*PCR Buffer, 1.5 mmol/L of Mg<2+>, 1 u of Tag enzyme, 1 u of glycosylation (UNG) enzyme, 0.1 mmol/L of deoxynucleotide triphosphate (dNTP), 10 pmol of specific primers of the lactobacillus fermentum and 10 pmol of probe. The fluorescence quantitative PCR method has the advantages of quick detection speed, high sensitivity and low cost.
Description
Technical field
The present invention relates to the quantitative fluorescent PCR method for quick of a kind of fermentation lactobacillus (Lactobacillus fermentum).
Background technology
Acid canned food (pH is below 4.5), the bacillus that microorganism mostly is acidproof, heat impedance is extremely strong that survives therein, coccus etc., their existence can cause the corruption of can usually.Because the sour environment that provides of acid canned food, wherein normally take the milk-acid bacteria of liking sour environment as dominant microflora, fermentation lactobacillus is wherein a kind of.The detection method that fermentation lactobacillus is commonly used at present is the anaerobism plating method, detects by an unaided eye after growing bacterium colony and selects suspicious bacterium colony, then carries out gramstaining and biochemical identification.The fermentation lactobacillus nutritional requirement is complicated, and cultural characters is more special, and the classic flat-plate incubation growth is slow, and separation and Culture needs 7～9d qualification cycle approximately, therefore sets up rapid detection and identifies that the method for inspection of fermentation lactobacillus is imperative.Fluorescent quantitative PCR technique is a kind of nucleic acid quantification technology that development in recent years is got up.This technology has been introduced fluorescently-labeled probe in the PCR reaction system, have the characteristics of high sensitivity, specificity and accuracy.Fluorescent quantitative PCR technique has been realized the leap of PCR from qualitative to quantitative, efficiently solves that PCR pollutes and to the quantitative inaccurate problem of template.At present, this technology has been widely used in eating source property microorganism detection field.Yet, the domestic report that is beneficial to fluorescence quantifying PCR method rapid detection fermentation lactobacillus that yet there are no so far.
Summary of the invention
The present invention is in order to solve the existing deficiency of prior art, the fluorescence quantifying PCR method of fast, the highly sensitive rapid detection fermentation lactobacillus of a kind of detection speed to be provided.
Technical solution of the present invention is: a kind of method of rapid detection fermentation lactobacillus is characterized in that having the following steps:
At first extract genomic dna and diluted for use that testing sample separates suspicious bacterium colony pure culture on the agar plate.
Then carry out the quantitative fluorescent PCR reaction, each quantitative fluorescent PCR reaction cumulative volume is 25 μ l, comprises the genomic dna of extraction, 2 μ l; 10 * PCR Buffer, 2.5 μ l; Mg
2+1.5mmol/L; Tag enzyme 1u; UNG enzyme 1u; DNTP 0.1mmol/L; Fermentation lactobacillus Auele Specific Primer, each 10pmol of probe; Add at last ddH
2O to 25 μ l; The program parameter that quantitative real time PCR Instrument is set is: 37 ℃ of 2min; 95 ℃ of denaturation 30s, 95 ℃ of sex change 5s, 60 ℃ of annealing 40s collect FAM fluorescence, 40 circulations simultaneously; 4 ℃ of preservations.
Wherein the fermentation lactobacillus Auele Specific Primer is:
L.fermenF-1：5’-CGTCGTTGACGAATACATCC-3’；
L.fermenR-2：5’-TCGATACGACCAGAAGCAAC-3’。
Specific probe is:
L.fermenF-3：5’-FAM-CAAGCCATTCATGATGCCTGTCG-3’。
After the quantitative fluorescent PCR reaction finished, fermentation lactobacillus can form typical " S " type amplification curve, and Ct value＜35.
The invention provides special primer L.fermenF-1, L.fermenR-2 and the probe L.fermenF-3 of amplification fermentation lactobacillus, thereby a kind of method of rapid detection fermentation lactobacillus be provided, compare with prior art and have following advantage:
1. detection speed is fast, directly utilizes the genomic dna that separates suspicious bacterium colony pure culture on the agar plate to detect, and saved gramstaining and biochemical identification step, and biochemical identification generally needs 24～72h.
2. cost is low, owing to omitted gramstaining and biochemical identification step, the expense of having saved gramstaining and biochemical reagents has reduced testing cost.
3. highly sensitive, because this technology has been introduced fluorescently-labeled probe in the PCR reaction system, improved the sensitivity that detects.
Embodiment
At first the 25g testing sample is got in aseptic technique, fully shreds, and adds to fill in the sterilising vessel of 225ml sterile saline, after carrying out 10 times of serial dilutions, select 2～3 suitable extent of dilution, each is applied on the MRS agar plate with 0.1mL, 36 ℃ ± 1 ℃, cultivate 48h under the amphimicrobian condition.3 of pickings or above suspicious bacterium colony are inoculated in and carry out pure culture on the MRS agar plate, and 36 ℃ ± 1 ℃, cultivate 48h under the amphimicrobian condition, extract genomic dna and diluted for use.
Then carry out the quantitative fluorescent PCR reaction, each quantitative fluorescent PCR reaction cumulative volume is 25 μ l, comprises the genomic dna of extraction, 2 μ l; 10 * PCR Buffer, 2.5 μ l; Mg
2+1.5mmol/L; Tag enzyme 1u; UNG enzyme 1u; DNTP 0.1mmol/L; Fermentation lactobacillus Auele Specific Primer, each 10pmol of probe; Add at last ddH
2O to 25 μ l; The program parameter that quantitative real time PCR Instrument is set is: 37 ℃ of 2min; 95 ℃ of denaturation 30s, 95 ℃ of sex change 5s, 60 ℃ of annealing 40s collect FAM fluorescence, 40 circulations simultaneously; 4 ℃ of preservations.
Wherein the fermentation lactobacillus Auele Specific Primer is:
L.fermenF-1：5’-CGTCGTTGACGAATACATCC-3’；
L.fermenR-2：5’-TCGATACGACCAGAAGCAAC-3’。
Specific probe is:
L.fermenF-3：5’-FAM-CAAGCCATTCATGATGCCTGTCG-3’
After the quantitative fluorescent PCR reaction finished, fermentation lactobacillus can form typical " S " type amplification curve, and Ct value＜35.
Claims (2)
1. the fluorescence quantifying PCR method of a rapid detection fermentation lactobacillus is characterized in that at first extracting genomic dna and the diluted for use that testing sample separates suspicious bacterium colony on the agar plate; Recycling fermentation lactobacillus genome conservative property dna sequence dna design Auele Specific Primer and probe; Then use this primer with probe the genomic dna that testing sample separates suspicious bacterium colony on the agar plate to be carried out fluorescent quantitative PCR and detection, wherein said fermentation lactobacillus Auele Specific Primer is:
L.fermenF-1：5’-CGTCGTTGACGAATACATCC-3’；
L.fermenR-2：5’-TCGATACGACCAGAAGCAAC-3’；
Specific probe is:
L.fermenF-3：5’-FAM-CAAGCCATTCATGATGCCTGTCG-3’。
2. the fluorescence quantifying PCR method of rapid detection fermentation lactobacillus according to claim 1, its application of sample parameter is: each quantitative fluorescent PCR reaction cumulative volume is 25 μ l, comprises the genomic dna of extraction, 2 μ l; 10 * PCR Buffer, 2.5 μ l; Mg
2+1.5mmol/L; Taq enzyme 1u; UNG enzyme 1u; DNTP 0.1mmol/L; Fermentation lactobacillus Auele Specific Primer, each 10pmol of probe add ddH at last
2O to 25 μ l; The program parameter that quantitative real time PCR Instrument is set is: 37 ℃ of 2min; 95 ℃ of denaturation 30s, 95 ℃ of sex change 5s, 60 ℃ of annealing 40s collect FAM fluorescence, 40 circulations simultaneously; 4 ℃ of preservations.
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Non-Patent Citations (4)
| Title |
|---|
| Comparison of partial tuf gene sequences for the identification of lactobacilli;Frederic Chavagnat,et al.;《FEMS Microbiology Letters》;20021106;第217卷;第177-183页，尤其是第177页第2栏第2段及第178页2.1节、2.4节及179页图1（a） * |
| Frederic Chavagnat,et al..Comparison of partial tuf gene sequences for the identification of lactobacilli.《FEMS Microbiology Letters》.2002,第217卷第177-183页. |
| 童睿等.食品中乳酸杆菌的实时荧光PCR的快速检测.《现代食品科技》.2008,第24卷(第1期),第86-88页. |
| 食品中乳酸杆菌的实时荧光PCR的快速检测;童睿等;《现代食品科技》;20080115;第24卷(第1期);第86-88页，尤其是第86页第1栏第2段及第2栏1.3.1节至第87页1.3.2.3节 * |
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