CN100395347C - Primer for detecting salmonella nucleotide fragment and probe sequence - Google Patents

Abstract

The present invention relates to a PCR amplification primer and a probe sequence for salmonella nucleotide fragments. A primer sequence comprises a primer sequence obtained in a region range of a primer pair (composed of an upstream primer SALF with a sequence of GCGGCGTTGGAGAGTGATA and a downstream primer SALR with a sequence of AGCAATGGAAAAAGCAGGATG), 10 basic groups (extended in the 5' end direction from the upstream primer SALF of the primer pair), 10 basic groups (extended in the 3' end direction from the upstream primer SALF), 10 basic groups (extended in the 3' end direction from the downstream primer SALR) and 10 basic groups (extended in the 5' end direction from the downstream primer SALR). The probe sequence comprises a probe sequence obtained in a region range of 10 basic groups (extended in the 3' end direction from a probe SAL-p1 with a sequence of CATTTCTTAAACGGCGGTGTCTTTCCCT) and 10 basic groups (extended in the 5' end direction).

Description

A kind of primer and probe sequence that is used to detect the Salmonellas nucleotide fragments

Technical field

The present invention relates to a kind of primer and probe sequence that is used to detect the Salmonellas nucleotide fragments.

Background technology

Salmonellas is pathogenic bacterium common in the import and export food, also is to cause poisoning by food and the The main pathogenic fungi of food origin disease, accounts for first of food poisoning in China.Salmonella food poisoning, except that the summer and autumn of main generation, all can cause that the food of poisoning is mainly meat, bird, eggs and milk the whole year, bean product and cake also take place sometimes.Except that food poisoning, it also can cause multiple human diseasess such as gastro-enteritis, microbemia, enteric fever.In 2002 the 25th of State Administration for Quality Supervision and Inspection and Quarantine and 26 commands clearly the regulation Salmonellas be essential items for inspection.Present detection to this bacterium, GB and the rower traditional flat board cultivation or the methods of integrated enzyme reaction (ELISA) of adopting more, these method stepss are loaded down with trivial details, waste time and energy, generally take 4-6 consuming time days at least, and because the influence of multiple interfering factors, the accuracy of detected result reduces easily, has brought very adverse influence for the import and export of food.Therefore, it is imperative to set up a kind of pathogenic bacterium detection method quicker, accurate, easy and simple to handle.

Domestic and international application mainly is divided three classes: regular-PCR technology, fluorescent PCR technology and biochip technology in the Protocols in Molecular Biology that foodborne bacterial pathogens detects at present.Method for gene chip detection efficiency height, but technology that is that all right is ripe, false positive rate and false negative rate all are difficult to control, and cost is higher, also is in conceptual phase at present.Regular-PCR method and technology maturation also is used for the detection of foodborne bacterial pathogens the earliest, but need carry out aftertreatment to the PCR product, very easily causes the PCR product pollution, and certain non-specific amplification is arranged.Fluorescent PCR is on the basis of regular-PCR, adds a specific fluorescent probe again in a pair of Auele Specific Primer of adding in amplification reaction system, uses the fluorescent PCR detector of monitoring in real time to detect the technology of target nucleotide sequences.Except the advantage with regular-PCR, it also has the following advantages: (1) specificity is stronger, and sensitivity is higher.Since used more one can with the fluorescent probe of template complementary pairing, improved specificity, and collected fluorescent signal by self-reacting device, avoided the subjectivity of artificial judgment, can further improve sensitivity again.(2) totally-enclosed reaction, online real-time monitoring fluorescence, aftertreatment that need not the PCR product is avoided polluting, and has guaranteed result's reliability.(3) data analysis is selected in the logarithmic phase of nucleic acid amplification, abandons the multifactor interferential end point analysis method that is subjected to of regular-PCR method, makes quantitatively more accurately and reliably.(4) can realize the two inspections of single tube or many inspections, also can design mark in the specific aim, monitoring extraction efficiency and get rid of inhibitor and disturb.(5) do not contact toxic reagent, operational safety.(6) help mass-producing, automatization and network management.(7) scope of application is wider, can detect the nucleic acid of any bacterium in theory.

Summary of the invention

The purpose of this invention is to provide a kind of primer and probe sequence that is used to detect the Salmonellas nucleotide fragments.

Based on above-mentioned purpose, the present invention by the following technical solutions:

The primer and the probe that are used to detect the Salmonellas nucleotide fragments comprise:

1. a primer that is used to detect the Salmonellas nucleotide fragments is right, it is characterized in that described primer is to being: by sequence is that the primer formed of the upstream primer SALF of GCGGCGTTGGAGAGTGATA and downstream primer SALR that sequence is AGCAATGGAAAAAGCAGGATG is right.

2. a probe that is used to detect the Salmonellas nucleotide fragments is characterized in that described probe SAL-pl sequence is CATTTCTTAAACGGCGGTGTCTTTCCCT.

Concrete principle of the present invention is to utilize Auele Specific Primer and a specificity fluorescent probe of a pair of target nucleotide sequences, adopt hot resistant DNA polymerase (Taq enzyme), four kinds of nucleotide monomer compositions such as (dNTP), and use the nucleic acid fragment amplification that round pcr is realized target nucleotide sequences.Employed probe is the oligonucleotide of two ends difference mark fluorescent reporter group (R) and fluorescent quenching group (Q).When probe is complete, the reporter group fluorescent signal emitted is absorbed by quenching group, and in the pcr amplification process, 5 ' end 5 prime excision enzyme activity of Taq enzyme is cut degraded with the fluorescent probe enzyme of specific combination on the target nucleotide fragment, the fluorescence report group is free in the reaction system, the shielding effect that has broken away from the fluorescent quenching group, the fluorescent signal of fluorescence report group just can by instrument detecting to, the variation of fluorescent signal amount is directly proportional with the amplified production amount, thereby judges the existence of target nucleotide sequences in the sample to be tested.

Description of drawings

Fig. 1 utilizes primer SALF/SALR and probe SAL-p1 to be detected the fluorescent PCR amplification figure of Salmonellas positive.

Embodiment

1. primer and probe design: by respectively all known salmonella gene group sequences being compared analysis, select the section (Salmonellas fimY gene) of no secondary structure and high conservative, design many to primer and probe, primer length is generally about 20 bases, between primer and primer in no complementary sequence.Optimum primer, probe combinations are as follows:

Upstream primer SALF:GCGGCGTTGGAGAGTGATA

Downstream primer SALR:AGCAATGGAAAAAGCAGGATG

Probe SAL-p1:CATTTCTTAAACGGCGGTGTCTTTCCCT

2. the foundation of reaction system and optimization: the target region template that is adopted in the foundation of reaction system and the optimization obtains with following method: get Salmonellas reference culture recovery back and cultivated 48 hours, 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 serial positive template, extract genomic nucleic acids respectively, carry out pcr amplification with the primer and the probe of the longest amplified fragments in the above-mentioned detection sequence area respectively again, and the template when getting wherein person between the Ct value 24-27 as reaction system optimization later on.

2.1 the optimization of primer concentration in reaction system, is done the primer concentration of Salmonellas to detect after the multiple proportions serial dilution from 0.1 μ mol/L to 0.8 μ mol/L respectively, compares by the analysis of test-results, determines that best primer final concentration is 0.2 μ mol/L.

2.2 under the constant prerequisite of the optimization of magnesium ion concentration other condition in reaction system, with MgCl ₂Concentration increase progressively with 0.5mmol/L from 1mmol/L to 2.5mmol/L, be magnesium ion concentration in the test kit reaction system through the selected 2.5mmol/L of repeated experiments repeatedly.

2.3Taq the optimization of archaeal dna polymerase (Taq enzyme) consumption is by comparing the optimization experiment result of Taq enzyme dosage (in the Unit of unit), selected 2U is as the consumption of Taq enzyme in the test kit reaction system.

2.4dNTPs the optimization of concentration detects by the dNTPs that uses different concns, selects the usage quantity of 0.2mmol/L as dNTPs in the test kit reaction system after the comprehensive assessment.

2.5 the optimization of concentration and probe concentration in reaction system, is done the concentration and probe concentration of Salmonellas to detect after the multiple proportions serial dilution from 0.05 μ mol/L to 0.2 μ mol/L respectively, compares by the analysis of test-results, determines that best probe final concentration is 0.1 μ mol/L.

Utilize above-mentioned primer and probe to carry out the foundation of reaction system, determine that at last the fluorescent PCR reaction system that adopts is 40 μ l systems, required each component and respective concentration see Table 1.

PCR reaction system after table 1 is optimized

Component	Final concentration
		10 * PCR reaction buffer	1×
Mg 2+Concentration	2.5mmol/L
		DNTPs (containing dUTP)	0.2mmol/L
The Taq enzyme	2U
		Primer (upstream)	0.2μmol/L
Primer (downstream)	0.2μmol/L
		Probe	0.1μmol/L
Template	2μl
		Moisturizing extremely	40μl

Annotate: a. at the fluorescent PCR reaction volume not simultaneously, each reagent should be adjusted in proportion.

B. the instrument difference of Shi Yonging should be done reaction parameter suitably to adjust.

3. the selection of instrument detecting passage: when carrying out the fluorescent PCR reaction, the collection of tackling reaction tubes fluorescent signal in the used instrument is provided with, and the fluorescence detection channel of selection is consistent with the fluorescence report group of probe institute mark.Concrete method to set up is different because of instrument, should be with reference to the instrument working instructions.

4.PCR it is as follows that condition is selected:

95 ℃ of 2min, 1 circulation;

95 ℃ of 5sec, 60 ℃ of 40sec, 40 circulations.

5. detection step:

(1) chooses primer and probe;

(2) prepare template to be measured, can adopt phenol-chloroform method to extract the genomic dna of Salmonellas in the sample of various sources;

(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;

(4) sense channel of selection instrument;

(5) go up machine testing.

6. embodiment

Choose primer to SALF/SALR and probe SAL-p1, with Salmonellas nutrient solution to be checked phenol-chloroform method extracting genomic dna.Concrete steps are as follows:

(1) Salmonellas enrichment liquid to be checked (about 1ml) is added in the centrifuge tube of 1.5ml, centrifugal 5 minutes of 12000rpm removes supernatant.

(2) add dna cleavage liquid 700ul, fully mixing is resuspended, and water-bath was boiled 5 minutes.

(3) add isopyknic phenol-chloroform (V/V=1: 1) solution, fully centrifugal behind the 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, the mixing that turns upside down, centrifugal 5 minutes of 13000rpm.

(6) use 70% alcohol flushing after abandoning supernatant, centrifugal 5 minutes of 13000rpm, the careful suction abandoned supernatant, and inversion is dried.

(7) in dried centrifuge tube, add the abundant mixing of 50ul DNA lysate, stand-by as dna profiling.

In 40ul fluorescent PCR reaction system, add the above salmonella gene group DNA 2ul that extracts, carry out fluorescent PCR according to aforementioned PCR reaction conditions and detect.After testing, then show positive amplification curve if contain Salmonellas in the nutrient solution to be checked, its detection sensitivity can reach 1000 copy/ml; Then do not have amplified signal if do not contain Salmonellas in the nutrient solution to be checked, point out above-mentioned primer having good sensitivity and specificity with probe.

7. advantage of the present invention:

(1) detection sensitivity of primer provided by the invention and probe can reach 1000 copy/ml, illustrates that it has good sensitivity.

(2) primer provided by the invention and probe without amplified signal, illustrate that it has good specificity for the detection sample standard deviation that does not contain salmonella.

(3) because the present invention adopts the endogenous gene fimY gene of salmonella as the genes of interest of amplification, avoided the generation of false negative result.

(4) because the present invention adopts Fluorescence PCR assay as detection method, whole reaction is all carried out in the reaction tube of sealing, has avoided other nucleic acid detection methods such as PCR-electrophoresis etc. to be easy to form Aerosol Pollution and causes false positive results; Because the PCR product is carried out Real-Time Monitoring, greatly saved monitoring time, saved manpower and materials.

＜110〉Shenzhen Taitai Genetic Engineering Co., Ltd.

＜120〉a kind of primer and probe sequence that is used to detect the Salmonellas nucleotide fragments

<140>CN200410051209.7

<141>2004-08-20

<160>3

<170>PatentIn version 3.3

<210>1

<211>19

<212>DNA

＜213〉artificial sequence

<400>1

gcggcgttgg agagtgata 19

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<400>2

agcaatggaa aaagcaggat g 21

<210>3

<211>28

<212>DNA

＜213〉artificial sequence

<400>3

catttcttaa acggcggtgt ctttccct 28

Claims (2)

1. a primer that is used to detect the Salmonellas nucleotide fragments is right, it is characterized in that described primer is to being: by sequence is that the primer formed of the upstream primer SALF of GCGGCGTTGGAGAGTGATA and downstream primer SALR that sequence is AGCAATGGAAAAAGCAGGATG is right.

2. a probe that is used to detect the Salmonellas nucleotide fragments is characterized in that described probe SAL-p1 sequence is CATTTCTTAAACGGCGGTGTCTTTCCCT.

Images