CN104498631B - Cause is laid eggs abnormal important virus multiple PCR detection primers and method - Google Patents

Abstract

The present invention provides one kind and causes lay eggs abnormal important virus multiple PCR detection primers and method, preparation is directed to 3 kinds of specific capture probes for causing the viral nucleic acids such as poultry egg laying extremely important viral egg-laying reduction syndrome virus, fowl tembusu virus and H9 subtype avian influenza virus, after hybridizing with corresponding viral nucleic acid, it is cyclized in the case where connecting enzyme effect, then enter performing PCR amplification by the universal detector primer of a pair of probe specificities to be detected, pathogenic infection is determined according to the amplified production size obtained.This method can be not only used for single viral detection, again can simultaneously 3 kinds of different viruses of antidiastole, the characteristics of with high efficiency, high specific, high sensitive, low cost, suitable egg fowl occur laying eggs exception when a large amount of clinical samples detection and analysis, be clearly to cause lay eggs the early stage rapid differential diagnosis of abnormal cause of disease and the important technical of analysis of molecular epidemiology.

Description

Cause is laid eggs abnormal important virus multiple PCR detection primers and method

Technical field

The present invention relates to cause lay eggs abnormal important virus multiple PCR detection primers and method, and in particular to a kind of ligase The cause of the dependence abnormal important virus multiple PCR that lays eggs differentiates detection method and its application, belongs to Preventive Veterinary Medicine field.

Background technology

In the last few years, China kind fowl and egg fowl in feeding process because epidemic disease cause to lay eggs exception the problem of it is more and more tight Weight, it is mainly shown as that egg production and Egg Quality are abnormal, as egg production is difficult to reach normal peak or declines suddenly, soft shell occurs Egg, husky shell egg, shell egg, lopsided egg and small-sized egg etc., have a strong impact on kind（Egg）The production performance of fowl, cause tremendous economic damage Lose.Now there are some researches show many cause of diseases can cause to plant（Egg）Fowl egg drop reduction, such as fowl tembusu virus（ATV）, egg drop reduction Syndrome virus（EDSV）, H9 subtype avian influenza virus (H9AIV), avian paramyxovirus 1（APMV-1）And salmonella etc. is more Kind bacterial pathogen.The epidemiology survey of laying poultry shows that more than 60% abnormal case of laying eggs is by three kinds of viruses One or two infection causes, and is clinically classified by caused many clinical symptoms after these three pathogen infections with lesion ten Seemingly, Chang Jifa or concurrent and easy mistaken diagnosis or delay treatment, so as to trigger even more serious economic loss.

Fowl tembusu virus belongs to the RNA virus of flaviviridae Flavivirus, is in April, 2010 in China Zhejiang, Jiangsu With the ground such as Fujian is newfound causes out laying duckses, kind duck to occur with rapid drawdown of laying eggs, the low dead acute height for principal character The cause of disease of contagious disease.Membrana follicularis hyperemia, the bleeding of laying poultry, ovarian follicle are mainly caused after virus infection laying poultry Denaturation, atrophy and scarring etc., the egg laying performance of strong influence poultry, laying rate of poultry decline reaches 30%- after 2 days after morbidity 70%, stop production within serious 7 ~ 14 days after morbidity, irreversibility stopping production occur in part cases of infection.To the end of the year 2014, China southeast There is the sick prevalence in coastal and surrounding area, and except laying duckses and chicken, goose, meat duck and sparrow etc. have infection to report, should Disease has seriously endangered the sustainable and healthy development to China's aviculture.

Egg-laying reduction syndrome virus belongs to the DNA virus of the type of aviadenovirus III, is to cause poultry in discovery in 1976 A kind of cause of disease of infectious disease characterized by egg drop reduction, mainly cause chicken group to lay eggs suddenly after virus infection and decline, soft shell Egg and the increase of lopsided egg etc., therefore egg drop syndrome 1976 is named as again.The chicken and duck of all ages and classes can infect, but young age Poultry does not show clinical symptoms.After laying poultry infects the virus, start to be with or without under general diarrhea, appetite during morbidity Drop and dispirited, subsequent eggshell colour fading, the soft-shelled egg that then rises, shell egg.The course of disease continues 4~10 weeks, egg drop reduction amplitude Up to 10%~40%, hatching of breeding eggs rate is reduced, and weak young bird is increased after going out shell, egg drop reduction continue 4~10 weeks it is latter as can recover normal.

H9 subtype avian influenza virus is the RNA virus of orthomyxovirus section, and now there are some researches show the virus infects laying poultry The harm such as laying rate, rate of fertilization and the incubation rate decline of poultry can equally be caused afterwards.When 20 ~ 30 week old kind fowl infect, meeting The respiratory symptom of appearance, while group decline occurs in egg production, egg production can decline the % of 30 % ~ 70, portion in 4 ~ 5 days The egg fowl of point case is even completely stopping to lay eggs.After 4 weeks, egg fowl slowly recovers to lay eggs, but does not reach the water before infection It is flat.

The PCR amplification techniques that ligase relies on are a kind of nucleic acid detection techniques mediated by ligase, and its operation principle is： The exogenous nucleic acid sequences artificial synthesized using one section utilize its both ends connection and nucleic acid array complementation to be checked as capture probe Target in oligonucleotide sequence, with testing sample nucleic acid hybridization, so as in nucleic acid chains to be checked formed one it is notched, Ring close from beginning to end, then the ring is closed to form a complete ring in connection enzyme effect；Finally use and be directed to the ring external source core The specific primer of acid sequence part enters performing PCR amplification under archaeal dna polymerase catalysis, reaches the purpose of detection testing gene.When When detecting multiple purpose nucleic acid sequences, it need to only add and be hybridized respective specific capture probe, finally with a pair of inspections Survey primer and enter PCR amplifications, you can reach the purpose of antidiastole.The technology does not expand nucleic acid to be checked directly, but passes through amplification The foreign gene hybridized with gene specific to be checked detects the purpose of nucleic acid to be checked to reach, and has both avoided the non-specific of detection Property, and because reducing primer quantity during PCR amplifications, detection efficiency and sensitiveness is guaranteed.

Therefore, the PCR amplification technique principles relied on according to ligase, establishing one kind can rapid differential diagnosis fowl Tan Busu disease Poison, egg-laying reduction syndrome virus, the multi-PCR detection method of H9 subtype avian influenza virus are laid eggs abnormal sick as laying poultry Making a definite diagnosis in time for cause of disease is highly effective when former routinely detection and epidemic disease happen suddenly, for the early stage for abnormal epidemic disease cause of disease of laying eggs The formulation and execution of diagnosis and prophylactico-therapeutic measures provide reliable technical support.

The content of the invention

The present invention provides cause and laid eggs abnormal important virus multiple PCR detection primers and method.This method is to be based on ligase Operation principle, hybridized with specific capture probe with corresponding viral nucleic acid, and be cyclized in the case where connecting enzyme effect, then should With a pair of universal primers to cyclisation, the capture probe for Different Kinds of Pathogens detect, according to obtain amplified production size Determine pathogenic infection.

The ligase refers to DNA Taq ligases.

It is abnormal important viral for egg-laying reduction syndrome virus, fowl tembusu virus that described 3 kinds cause egg fowl to be laid eggs And H9 subtype avian influenza virus, its specific sequence capture probe is respectively such as Seq ID No.1, Seq ID No.2 and Seq Shown in ID No.3.

For the universal detector primer sequence such as Seq ID No.4 and Seq ID No.5 institutes of 3 kinds of viral nucleic acid capture probes Show.

It detects program and mainly divided 3 stages, and the first stage is denaturation stage, and 95 DEG C maintain 5 min；Second stage is company The capture probe of enzyme mediation and hybridization and the circularizing probes stage of template are connect, 95 DEG C of 50 s, 55 DEG C ~ 63 DEG C 5-10 min enter 6 circulations of row；Phase III is PCR detection-phases 95 DEG C of 30 s, 52 DEG C ~ 56 DEG C 30 s, and 72 DEG C of 30 s carries out 25 Circulation, last 72 DEG C of 10 min terminate to react.

To obtain foregoing invention purpose, the technical solution adopted by the present invention is：Multiplexed PCR amplification side dependent on ligase Method, comprise the following steps：

1st, the preparation of etiology nucleic acid specific probe：According to announced egg-laying reduction syndrome virus in GenBank The hemagglutinin gene of 100kd GFPs, the capsid protein gene of fowl tembusu virus and H9 subtype avian influenza virus is protected Defending zone sequence conservation and enhanced green fluorescence protein（EGFP）The partial sequence of gene, design 3 pairs of primers, this 3 pairs of primers It is respectively the complementary oligonucleotide acid sequence of 3 kinds of etiology nucleic acid sequences above to be characterised by 5` ends, and 3` ends are foreign gene（This hair It is enhanced green fluorescent protein gene in bright）Nucleotide sequence.Using green fluorescent protein plasmid as template, with this 3 pairs of primers Standard PCR amplification is carried out respectively, and viral nucleic acid specificity capture is can be used as after 3 kinds of purified recovery of amplified production of acquisition and is visited Pin.

2nd, the design of universal detector primer：According to the consensus sequence of 3 kinds of cause of disease capture probes（Foreign gene part, that is, increase Strong type green fluorescence protein gene）A pair of reverse primers are designed, this is characterised by itself and cause of disease purpose to be detected to primer Gene affiliation is remote, after the specific enhanced green fluorescent protein gene hybridization with the capture probe that has been cyclized, Archaeal dna polymerase effect is lower to start PCR amplification procedures.

3rd, the foundation and optimization of multi-PCR detection method：

（A）The configuration of multi-PRC reaction system, the multi-PRC reaction system amount to 30 μ L, including dNTPs 0.3 μ L, 10 × Taq the DAN polymerases of 3 μ L, Taq DNA ligase of mM, 10 × Taq DNA connections enzyme reaction buffer solution 0.5 The μ L of reaction buffer 3（Containing Mg²⁺）, the μ L of Taq archaeal dna polymerases 0.50, each 8 pmol of universal detector primer, 3 kinds of captures Probe and each 2 μ L of DNA or cDNA solution, add deionized water to complement to 30 μ L, in addition configuration it is a without sample DNA or CDNA reaction solution does negative control.

（B）All reactions of response procedures of multiplex PCR are completed in same reaction tube, mainly point 3 stages, the One stage was denaturation stage, and 95 DEG C maintain 5 min；Second stage is capture probe and the hybridization and spy of template of ligase mediation Needle ring stage, 95 DEG C of 50 s, 55 DEG C ~ 63 DEG C 5-10 min carry out 6 circulations；Phase III is PCR detection-phases 95 DEG C 30 s, 52 DEG C ~ 56 DEG C 30 s, 72 DEG C of 30 s carry out 25 circulations, and last 72 DEG C of 10 min terminates to react.Amplification Electrophoresis showed testing result is carried out after end immediately.The specificity of PCR detections can also be by will send after PCR primer purifying recovery Raw work Shanghai bioengineering Co., Ltd sequence verification.

（C）In the technical scheme of the optimization optimization of detection technique, using above-mentioned 3 kinds of capture probes and the core of measuring samples During acid hybridization, the dosage of each probe is 8-12 pmoL in every 30 μ L PCR reaction systems.

In the technical scheme of optimization, when the universal primer of application detects the capture probe of cyclisation, every 30 μ L PCR reactants The dosage of each probe is 7-10 pmoL in system.

The remarkable advantage of the present invention：

According to the operation principle of ligase, the present invention establishes the cause that a kind of ligase relies on and laid eggs abnormal important disease first Malicious multiplex PCR detection technique, it is specific to be applied to cause the extremely important viral egg-laying reduction syndrome virus of poultry egg laying, fowl The quick discriminating of tembusu virus and H9 subtype avian influenza virus.This method can be not only used for single viral detection, again can be simultaneously Differentiate 3 kinds of different viruses, there is the characteristics of high efficiency, inexpensive.

In the present invention involved viral nucleic acid specificity capture probe be with detected 3 kinds of nucleic acid sequence parents The all far green fluorescence protein gene fragment of edge relation is skeleton, and one section and viral nucleic acid to be detected are respectively connected at fragment both ends It is prepared after the complementary oligonucleotide sequence of sequence.Pass through the target gene complementary series specificity capture purpose at probe both ends Viral gene, and middle green fluorescence protein gene the fragment then consensus sequence as 3 kinds of probes and the target that is detected as PCR Gene.Therefore, the problem of both having avoided the mutual interference amplification efficiency of more primers in traditional multiple PCR technique, but it is because logical The non-specificity for expanding and detection being avoided with the foreign gene of gene recombination to be checked is crossed, improves the accuracy and sensitivity of detection Property.

Brief description of the drawings

Fig. 1 is the electrophoretogram of pcr amplification product in embodiment.Each swimming lane is represented as respectively in figure：M、DNA Marker (DL2000);1st, Egg Drop syndrome virus；2nd, fowl tembusu virus；3rd, H9 subtype avian influenza virus；4th, Egg Drop syndrome virus + fowl tembusu virus；5th, Egg Drop syndrome virus+H9 subtype avian influenza virus；6th, fowl tembusu virus+H9 subtype avian influenzas disease Poison；7th, Egg Drop syndrome virus+fowl tembusu virus+H9 subtype avian influenza virus；8th, negative control.

Embodiment

Embodiment 1

The preparation of virus-specific capture probe

It is used to detect 3 kinds of viral specific capture probes for causing poultry egg laying abnormal in the present invention, is by as follows Method derives what is obtained：

1.1st, according to announced egg-laying reduction syndrome virus in GenBank（EDSV）100kd genes, fowl Tan Busu The capsid protein gene of viral (ATV) and the hemagglutinin gene conserved region sequence of H9 subtype avian influenza virus (H9AIV) are protected Defending zone and enhanced green fluorescence protein（EGFP）Partial sequence, design 3 pairs of primers and be used to prepare capture probe（Primer information It see the table below 1）.

1.2nd, with green fluorescent protein plasmid（PIRES2-EGFP, Takara Bioisystech Co., Ltd, Dalian）As Template, 3 pairs of primers expand more than respectively.PCR reaction systems are 50 μ L：The μ L of 10 x FastPfu Buffer 5, FastPfu DNA polymerase 1 μL（2.5 u/μL）, the μ L of dNTP (10 mM) 1,10 primers each 1 μ L, the μ of template 2 L, with deionized water added body product to 50 μ L.Response procedures are as follows：94 DEG C of min of pre-degeneration 3, by 94 DEG C of 20 s, 55 DEG C 30 s, 72 DEG C of 1min carry out 35 circulations, and 10 min of last 72 DEG C of extensions terminate to react, and take 5 μ L products through 1.0 % fine jades Sepharose electrophoresis detection amplification, viral nucleic acid specificity capture probe is can be used as after the purified recovery of amplified production. To verify the correctness of these probes, purifying recovery probing pin clone can be entered flush end cloning vector pEASY-Blunt, sent in raw work Marine growth Engineering Co., Ltd is sequenced, and the sequence of 3 kinds of probes is respectively such as Seq ID No.1, Seq ID No.2 and Seq ID Shown in No.3.

Embodiment 2：

Ligase relies on the foundation of PCR detection method

2.1 according to the nucleotide sequences of acquired 3 kinds of viral capture probes, design a pair of reverse universal detector primers, on Trip primer is Seq ID No.4（rEGFPF）:5 '-CTCGGCGCGGGTCTTGTAGTT -3 ', anti-sense primer are Seq ID No.5（rEGFPR）:5 '-CTCGGCGCGGGTCTTGTAGTT -3 ', different virus nucleic acid is directed to primer amplification using this , cyclisation probe can obtain different size of nucleic acid fragment.

2.2 collection performances are laid eggs, and abnormal duck liver is dirty or fallopian tubal, after being homogenized according to a conventional method, with containing dual anti-sterile PBS Buffer solution (pH 7.2) carries out 1:3 dilutions, are divided into 2 parts, after multigelation 3 times, take 1 part to carry out 8 000 r min-1 centrifugations 10min, take supernatant DNR/RNA extraction kit（QIAGEN companies）STb gene and RNA are extracted respectively, with reference to kit Operational manual is carried out.The RNA sample obtained is extracted to grasp by Reverse transcription kit (Ivitrigen companies) Book is explained, obtains cDNA with hexabasic base random primed reverse transcription, the DNA that cDNA and extraction obtain is carried out respectively as next step The template of PCR amplifications.

Measuring samples DNA or the cDNA template of above-mentioned preparation, the capture probe of gel extraction and universal detector primer is taken to enter The PCR detection and analysis that row ligase relies on.PCR reaction systems are 30 μ L：Including the mmolL of dNTPs 0.3^-1, 10×Taq μ L, the Taq DNA ligases of DNA connections enzyme reaction buffer solution 3（5U/ µL）0.5 μ L, 10 × Taq DAN polymeric enzyme reactions The μ L of buffer solution 3（Containing Mg²⁺）, Taq archaeal dna polymerases（5U/ µL）0.50 μ L, universal detector primer each 20 pmol, 3 kinds Capture probe and each 2 μ L of DNA or cDNA solution, 30 μ L are complemented to deionized water.Response procedures are as follows：95℃ 5 min；95 DEG C of 50 s, 55 DEG C ~ 63 DEG C 7 min carry out 6 circulations；95 DEG C of 30 s, 53 30 s, 72 DEG C of 30 s enter 25 circulations of row, last 72 DEG C of 10 min terminate to react.Take the μ LPCR amplified productions of product 5 electric on 1.0% Ago-Gel Swimming, electrophoretic voltage are set as 5V/cm.When sample electrophoresis to appropriate location are, with ultraviolet gel imaging system（Bio-rad, Gel Doc 2000）Observe and record result（See Fig. 1）.The length of amplified fragments is respectively Egg Drop syndrome virus 436bp, fowl after electrophoresis Tembusu virus 480bp and H9 subtype avian influenza virus 681bp.It is made whether that infection is corresponding according to above amplified fragments are whether there is Virus judges.

The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, it should all belong to the covering scope of the present invention.

SEQUENCE LISTING

<110>Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute

<120>Cause is laid eggs abnormal important virus multiple PCR detection primers and method

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Claims (2)

1. the probe for the extremely important virus multiple PCR detection method of poultry egg laying, it is characterised in that：It is described specific to catch Probe sequence is obtained respectively as shown in Seq ID No.1, Seq ID No.2 and Seq ID No.3.

2. probe as claimed in claim 1 is preparing the reagent for the extremely important virus multiple PCR detection method of poultry egg laying In application, it is characterised in that：The detection method hybridizes using virus-specific probe with corresponding viral nucleic acid to be cyclized Afterwards, enter performing PCR by the universal detector primer of probe specificity to expand, reach while detect 3 kinds and cause poultry egg laying extremely sick Former purpose.