CN101294223B - Primer and probe sequence for testing type II pig circular ring virus nucleotide fragment - Google Patents

Abstract

The invention relates to a primer sequence and a probe sequence used for detecting ribonucleotide fragments of type 2 porcine circovirus. The primer sequence comprises a primer pair composed of an upstream primer PCV II pf517 and a downstream primer PCV II pr615, wherein, the sequence of the upstream primer PCV II pf517 is AAATCTCATCATGTCCACCGC, and the sequence of the downstream primer PCV II pr615 is GCTACCGTTGGAGAAGGAAAA. The probe sequence comprises probe PCV II pb585, the sequence of which is TTCAACACCCGCCTCTCCCGCA.

Description

A kind of primer and probe sequence that is used to detect II type pig circular ring virus nucleotide fragment

Technical field

The present invention relates to a kind of primer and probe sequence that is used to detect II type pig circular ring virus nucleotide fragment.

Background technology

Pig circular ring virus (porcine circovirus is hereinafter to be referred as PCV) is the sub-thread ring-type minus-strand dna virus of no cyst membrane, and the about 17-20nm of virus particle diameter is one of animal virus of known minimum so far.PCV belongs to PCV-II section (Circoviridae) PCV-II and belongs to the member, according to its antigenicity and genomic constitution difference, is divided into 2 kinds of genotype or claims serotype, be i.e. I type PCV (being called for short PCV1) and II type PCV (being called for short PCV2).PCV1 is virus of coming out as a kind of separated from contaminants from pig continuous cell line PK-15 first in 1974, and experimentation on animals shows this virus no pathogenicity, extensively is present in each histoorgan in the normal pig body.And the pmws of PCV2 and generation in recent years (postweaning multisystemicwasting syndrome, PMWS) closely related.The generation of this disease and spread and to have caused enormous economic loss for world's pig industry.This disease with gradually become thin, expiratory dyspnea, lymphadenectasis, dysplasia and ochrodermia etc. be principal character.Yet these symptoms are not that PCV2 infection back is peculiar, other many pathogen infections also can cause similar symptom, as porcine reproductive and respiratory syndrome virus (porcinereproductive and respiratory syndrome virus, PRRSV) infect, therefore must adopt suitable detection method to determine whether these symptoms infect relevant with PCV2.

Along with going deep into to PCV2 research, increasing data shows, the numerous disease that takes place in PCV2 and the swinery is closely related, comprises that the scorching nephrotic syndrome of PMWS, pigskin, breeding difficulty, A2 type are congenitally trembled, sick compound the levying and pig hyperplasia and necrotizing pneumonia etc. of porcine respiratory.

At present, there is virus to separate, indirect immunofluorescence antibody method (IFA), E11SA, in-situ nucleic acid hybridization (ISH), immunohistochemical method (IHC) and PCR etc. to PCV2 detection method commonly used.It is that the laboratory is made a definite diagnosis PCV-2 and infected sensitive method that virus is separated, but because PCV2 does not produce pathology on the PKI5 cell, virus separate length consuming time and difficulty big.Though in situ hybridization has very high sensitivity, to operate loaded down with trivial detailsly, cost is higher, and the cycle is long, and having relatively high expectations to pathological material of disease.E11SA is highly sensitive, but the result judges the influence that is subject to subjective factor, the conventional PCR detection method of widespread use at present is simply quick, sensitive, but occur crossed contamination easily, be easy to generate false positive, and because the extensive existence of PCV2, and virus quantity exists significantly differently in the swinery of clinical onset and inapparent infection, and conventional PCR is difficult to play the effect that real-time quantitative detects.Therefore, it is imperative to set up a kind of quicker, accurate, easy and simple to handle detection method.

Real-time quantitative PCR is the very fast a kind of detection method of development in recent years, it is on the basis of regular-PCR, add a specific fluorescent probe again when in amplification reaction system, adding a pair of Auele Specific Primer, use 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, need not its pcr amplification of PCR and the whole process of product analysis all under the single tube sealing condition, carry out, the Real-time and Dynamic Detection and the result that have realized amplified production analyze automatically, avoided aftertreatment to amplified production, avoid the laboratory crossed contamination effectively, can obtain the result in the short period of time simultaneously.(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 virus 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 II type pig circular ring virus nucleotide fragment.

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

The primer and the probe sequence that are used to detect II type pig circular ring virus nucleotide fragment comprise: by upstream primer PCV II pf517 sequence is that AAATCTCATCATGTCCACCGC and downstream primer PCV II pr615 sequence are that the primer formed of GCTACCGTTGGAGAAGGAAAA is right.Probe PCV II pb585 sequence is TTCAACACCCGCCTCTCCCGCA.

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

Accompanying drawing: utilize primer PCV II pf517/pr615 and probe PCV II pb585 to be detected the fluorescent PCR amplification figure of II type pig circular ring virus positive.

Embodiment

1. primer and probe design: by respectively all known II type pig circular ring virus genome sequences being compared analysis, select the section of no secondary structure and high conservative, design manyly to primer and probe, primer length is generally about 20 bases, no complementary sequence between primer and in the primer.Optimum primer, probe sequence make up as follows:

Upstream primer PCV II pf517:AAATCTCATCATGTCCACCGC

Downstream primer PCV II pr615:GCTACCGTTGGAGAAGGAAAA

Probe PCV II pb585:TTCAACACCCGCCTCTCCCGCA

2. the foundation of reaction system and optimization:

2.1 the foundation of positive template: the target region template obtains with following method: the II type pig circular ring virus 2 poison strain that utilizes deactivation is as sample to be checked, extracts virus genom DNA with the extracting method of phenol chloroform, be stored in after the packing-20 ℃ standby.

2.2 the optimization of primer concentration is in reaction system, the primer concentration of II type pig circular ring virus 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.

2.4 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.

2.5Taq 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.

2.6dNTPs 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.

2.7 the optimization of concentration and probe concentration is in reaction system, the concentration and probe concentration of II type pig circular ring virus 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
		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 ℃ 2 minutes, 1 circulation; 95 ℃ 5 seconds, 60 ℃ 40 seconds, 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 II type pig circular ring virus 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 PCV II pf517/pr615 and probe PCV II pb585, with sample tissue to be checked phenol-chloroform method extracting viral DNA.Concrete steps are as follows:

(1) clip 50-100mg flesh tissue sample adds in the mortar that 500ul Hanks liquid is housed and slowly is ground into homogenate, gets homogenate and is for further processing as sample.If sample is a blood, gets serum and be for further processing as sample.

(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, 13000 rev/mins centrifugal 5 minutes.

(4) supernatant liquor is moved in the centrifuge tube of another 1.5ml, add isopyknic chloroform, mixing, 13000 rev/mins are centrifugal 5 minutes.

(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, 13000 rev/mins are centrifugal 5 minutes.

(6) use 70% alcohol flushing after abandoning supernatant, 13000 rev/mins centrifugal 5 minutes, the careful suction abandoned supernatant, 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 II type pig circular ring virus 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 II type pig circular ring virus 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 II type pig circular ring virus in the nutrient solution to be checked, point out above-mentioned primer having good sensitivity and specificity with probe.

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 do not have amplified signal for the detection sample standard deviation that does not contain II type pig circular ring virus, illustrate that it has good specificity.

(3) because the present invention compares analysis to all known II type pig circular ring virus genome sequences respectively, select the section of no secondary structure and high conservative to carry out the design of primer and probe, 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?LISTING

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

＜120〉a kind of primer and probe sequence that is used to detect II type pig circular ring virus nucleotide fragment

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<170>PatentIn?version3.3

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＜213〉artificial sequence

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＜213〉artificial sequence

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Claims (1)

1. one kind is used to detect the primer of II type pig circular ring virus nucleotide fragment and the combined prod of probe, it is characterized in that described primer is: by upstream primer PCV II pf517 sequence is that AAATCTCATCATGTCCACCGC and downstream primer PCV II pr615 sequence are that the primer formed of GCTACCGTTGGAGAAGGAAAA is right; Described probe PCV II pb585 sequence is TTCAACACCCGCCTCTCCCGCA.

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