CN100395350C - Prime and probe sequence for detecting nucleotide fregment of comma bacillus - Google Patents

Abstract

The present invention relates to a PCR amplification primer and a probe sequence for comma bacillus nucleotide fragments. A primer sequence comprises a primer sequence obtained in a region range of a primer pair (composed of an upstream primer F1253 with a sequence of GCTTTATTGTTCGATGCGTTAAAC and a downstream primer R1089 with a sequence of GATGCCAAAATTGTGCGTATCA), 10 basic groups (extended in the 5' end direction from the position of the upstream primer F1253 of the primer pair), 10 basic groups (extended in the 3' end direction from the position of the upstream primer F1253), 10 basic groups (extended in the 3' end direction from the position of the downstream primer R1089) and 10 basic groups (extended in the 5' end direction from the position of the downstream primer R1089). 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 Pb1218 with a sequence of TCTTGGGCAATCGCATCGGTTGA) and 10 basic groups (extended in the 5' end direction).

Description

A kind of primer and probe sequence that is used to detect nucleotide fregment of comma bacillus

Technical field

The present invention relates to a kind of primer and probe sequence that is used to detect nucleotide fregment of comma bacillus.

Background technology

Vibrio cholerae is a common pathogenic bacteria in food, is to cause poisoning by food and the The main pathogenic fungi of food origin disease.Cholera is the acute infectious disease that is caused by vibrio cholerae (Vibrio cholerae), and it falls ill anxious, propagates soon, involves widely, and harm is serious.It is defined as one of transmissible disease that must international quarantine by the World Health Organization, China classifies it as should implement " mandatory administration " category A infectious disease in the law on the prevention and control of infectious diseases center, also is to plant in international quarantine transmissible disease the most serious a kind of when first three.Cholera is the infectious intestinal disease of peroral infection, Chang Jingshui, food, life contact and fly etc. and propagate.Water-borne transmission is topmost route of transmission, and is all previous more popular or break out how contaminated relevant with water body.The characteristics of water-borne transmission are often to present to break out, and patient is many to distribute along contaminated water body.Severe cholera patient's main clinical manifestation is violent diarrhoea, vomiting, dehydration, circulatory failure and metabolic acidosis etc.As rescue untimely or improper, can be dead in many hours a few hours to ten in morbidity back.Under natural situation, the mankind are unique susceptible persons of vibrio cholerae.In the popular district of region, except that patient, the symptomless infection person also is important contagium.The route of transmission mainly is to take in by water source that pollutes or food per os, and interpersonal direct propagation is uncommon.In 2002 the 25th of State Administration for Quality Supervision and Inspection and Quarantine and 26 commands clearly the regulation vibrio cholerae 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 nucleotide fregment of comma bacillus.

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

The primer and the probe sequence that are used to detect nucleotide fregment of comma bacillus comprise:

1. a primer that is used to detect nucleotide fregment of comma bacillus is right, it is characterized in that described primer is to being: by sequence is that the primer formed of the upstream primer F1253 of GCTTTATTGTTCGATGCGTTAAAC and downstream primer R1089 that sequence is GATGCCAAAATTGTGCGTATCA is right.

2. a probe that is used to detect nucleotide fregment of comma bacillus is characterized in that described probe Pb1218 sequence is TCTTGGGCAATCGCATCGGTTGA.

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 F1253/R1089 and probe Pb1218 to be detected the fluorescent PCR amplification figure of vibrio cholerae positive.

Embodiment

1. primer and probe design: by respectively all known cholera vibrio gene group sequences being compared analysis, select the section (vibrio cholerae hlyA 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 sequence make up as follows:

Upstream primer F1253:GCTTTATTGTTCGATGCGTTAAAC

Downstream primer R1089:GATGCCAAAATTGTGCGTATCA.

Probe Pb1218:TCTTGGGCAATCGCATCGGTTGA

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 vibrio cholerae 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 vibrio cholerae 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 vibrio cholerae 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 vibrio cholerae 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 F1253/R1089 and probe Pb1218, with vibrio cholerae nutrient solution to be checked phenol-chloroform method extracting genomic dna.Concrete steps are as follows:

(1) vibrio cholerae 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 cholera vibrio 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 vibrio cholerae 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 vibrio cholerae 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 comma bacillus.

(3) because the present invention adopts the endogenous gene hlyA of comma bacillus 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.

Sequence table

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

＜120〉a kind of primer and probe sequence that is used to detect nucleotide fregment of comma bacillus

<160>3

<170>PatentIn version 3.3

<210>1

<211>24

<212>DNA

＜213〉artificial sequence

<400>1

gctttattgt tcgatgcgtt aaac 24

<210>2

<211>22

<212>DNA

＜213〉artificial sequence

<400>2

gatgccaaaa ttgtgcgtat ca 22

<210>3

<211>23

<212>DNA

＜213〉artificial sequence

<400>3

tcttgggcaa tcgcatcggt tga 23

Claims (2)

1. a primer that is used to detect nucleotide fregment of comma bacillus is right, it is characterized in that described primer is to being: by sequence is that the primer formed of the upstream primer F1253 of GCTTTATTGTTCGATGCGTTAAAC and downstream primer R1089 that sequence is GATGCCAAAATTGTGCGTATCA is right.

2. a probe that is used to detect nucleotide fregment of comma bacillus is characterized in that described probe Pb1218 sequence is TCTTGGGCAATCGCATCGGTTGA.

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