CN100386442C

CN100386442C - Prime and probe sequence for detecting nucleotide fregment of 01 Group comma bacillus

Info

Publication number: CN100386442C
Application number: CNB2005101208953A
Authority: CN
Inventors: 肖性龙; 张经纬
Original assignee: SHENZHEN TAITAI GENETIC ENGINEERING Co Ltd
Current assignee: SHENZHEN TAITAI GENETIC ENGINEERING Co Ltd
Priority date: 2005-12-15
Filing date: 2005-12-15
Publication date: 2008-05-07
Anticipated expiration: 2025-12-15
Also published as: CN1831142A

Abstract

The present invention relates to a PCR amplification primer and a probe sequence for 01 group 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 F605 with a sequence of CACGCCATTGAAGGTTATGTCTC and a downstream primer R703 with a sequence of AAATTGATGTCGATAGCGGTAGATTA), 10 basic groups (extended in the 5' end direction from position the upstream primer F605), 6 basic groups (extended in the 3' end direction from the position of the upstream primer F605 of the primer pair), 6 basic groups (extended in the 3' end direction from the position of the downstream primer R703) and 10 basic groups (extended in the 5' end direction from the position of the downstream primer R703). The probe sequence comprises a probe sequence obtained in a region range of 6 basic groups (extended in the 3' end direction from a probe Pb645 with a sequence of CCGCCTGCTCAGCAAAAGTATTCATCA) and 10 basic groups (extended in the 5' end direction).

Description

A kind of primer and probe sequence that is used to detect O1 group cholera vibrio nucleotide fragments

Technical field

The present invention relates to a kind of primer and probe sequence that is used to detect O1 group cholera vibrio nucleotide fragments.

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.Vibrio cholerae has heat-stable O antigen and heat labile H antigen.According to O antigen difference, now existing 155 serogroupss, wherein O1 group, O139 group cause cholera, the O1 group cholera vibrio infects can be from asymptomatic or light-duty lethality diarrhoea of suffering from diarrhoea serious.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 O1 group cholera vibrio nucleotide fragments.

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

The primer and the probe sequence that are used to detect O1 group cholera vibrio nucleotide fragments comprise:

1. a primer that is used to detect O1 group cholera vibrio 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 F605 of CACGCCATTGAAGGTTATGTCTC and downstream primer R703 that sequence is AAATTGATGTCGATAGCGGTAGATTA is right.

2. a probe that is used to detect O1 group cholera vibrio nucleotide fragments is characterized in that described probe Pb645 sequence is CCGCCTGCTCAGCAAAAGTATTCATCA.

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 F605/R703 and probe Pb645 to be detected the fluorescent PCR amplification figure of O1 group cholera vibrio positive.

Embodiment

1. primer and probe design: by respectively all known O1 group cholera vibrio genome sequences being compared analysis, select the section (O1 group cholera vibrio rfbM 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 F605:CACGCCATTGAAGGTTATGTCTC

Downstream primer R703:AAATTGATGTCGATAGCGGTAGATTA

Probe Pb645:CCGCCTGCTCAGCAAAAGTATTCATCA

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 O1 group cholera vibrio 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 is in reaction system, the primer concentration of O1 group cholera vibrio is done to detect after the multiple proportions serial dilution from 0.1 μ mol/L to 0.8 μ mol/L respectively, analysis by test-results is compared, and 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 is in reaction system, the concentration and probe concentration of O1 group cholera vibrio is done to detect after the multiple proportions serial dilution from 0.05 μ mol/L to 0.2 μ mol/L respectively, analysis by test-results is compared, and 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
Component	Final concentration	10 * PCR reaction buffer	1×
Mg ²⁺Concentration	2.5mmol/L	10 * PCR reaction buffer	1×
Mg ²⁺Concentration	2.5mmol/L	DNTPs (containing dUTP)	0.2mmol/L
The Taq enzyme	2U	DNTPs (containing dUTP)	0.2mmol/L
The Taq enzyme	2U	Primer (upstream)	0.2μmol/L
Primer (downstream)	0.2μmol/L	Primer (upstream)	0.2μmol/L
Primer (downstream)	0.2μmol/L	Probe	0.1μmol/L
Template	2μl	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 O1 group cholera vibrio 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 F605/R703 and probe Pb645, with O1 group cholera vibrio nutrient solution to be checked phenol-chloroform method extracting genomic dna.Concrete steps are as follows:

(1) O1 group cholera vibrio enrichment liquid (about 1ml) to be checked 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 O1 group cholera vibrio genomic 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 the O1 group cholera vibrio 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 the O1 group cholera vibrio 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 the O1 group cholera vibrio.

(3) because the present invention adopts the endogenous gene rfbM of O1 group cholera vibrio 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 O1 group cholera vibrio nucleotide fragments

<160>3

<170>Patent In version 3.3

<210>1

<211>23

<212>DNA

＜213〉artificial sequence

<400>1

cacgccattg aaggttatgt ctc 23

<210>2

<211>26

<212>DNA

＜213〉artificial sequence

<400>2

aaattgatgt cgatagcggt agatta 26

<210>3

<211>27

<212>DNA

＜213〉artificial sequence

<400>3

ccgcctgctc agcaaaagta ttcatca 27

Claims

1. a primer that is used to detect 01 group cholera vibrio 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 F605 of CACGCCATTGAAGGTTATGTCTC and downstream primer R703 that sequence is AAATTGATGTCGATAGCGGTAGATTA is right.

2. a probe that is used to detect 01 group cholera vibrio nucleotide fragments is characterized in that described probe Pb645 sequence is CCGCCTGCTCAGCAAAAGTATTCATCA.