CN101921869B - Method for identifying virulent strain and attenuated strain in Newcastle disease virus by pyrosequencing technology - Google Patents

Abstract

The invention belongs to the animal pathogen detection technical field, and relates to a method for identifying a virulent strain and an attenuated strain in a Newcastle disease virus by a pyrosequencing technology. The method comprises the following steps: firstly carrying out primer design to discriminate a specific primer and a pyrosequencing primer for the virulent strain and the attenuated strain in the Newcastle disease virus; extracting ribonucleic acid from a sample to be tested, carrying out reverse transcription-polymerase chain reaction (RT-PCR) for amplification by using the specific primer, and then carrying out agarose gel electrophoresis for detecting the amplified product; if the amplified fragment length of the specific primer is 268bp, preparing a pyrosequencing single-strand template for pyrosequencing reaction; and finally judging whether the sample contains the virulent strain or the attenuated strain in the Newcastle disease virus according to an amplification result and a pyrosequencing result. The method absorbs the advantages of RT-PCR and DNA sequencing technologies and meets the need for molecular diagnosis by specific sequence determination, thus achieving reliable identification result of the virulent strain or the attenuated strain in the Newcastle disease virus; and the method is fast, convenient and accurate.

Description

The tetra-sodium sequencing technologies is differentiated the method for NDV virulent strain and low virulent strain

Technical field:

The invention belongs to the detection technique field of animal pathogenic, relate to the method that a kind of employing tetra-sodium order-checking (Pyrosequencing) technology is differentiated NDV virulent strain and low virulent strain.

Background technology:

Newcastle disease (Newcastle disease; ND) be the strong communicable disease of present serious harm livestock industry and human health; Two sick propagation all can cause respiratory system disease and the general septicemia of bird, and nearly all wild and domestic bird all can infect.Now, newcastle disease (ND) is classified as the category-A transmissible disease by International Office of Epizootics (OIE), and China Ministry of Agriculture also is decided to be one type of animal epidemic to them.Newcastle disease be by NDV (Newcastle disease virus, NDV) cause bird with expiratory dyspnea, the yellow-green colour of having loose bowels just, nervous function disorder and mucous membrane, serous coat is hemorrhage is height contact, the acute septic transmissible disease of principal character.Though NDV has only a serotype, at present the biological characteristicses such as virulence between tens of kinds of strains are widely different in the world.Generally can NDV be divided into anaphylactic type strain, middle hair style strain and delayed type strain by traditional pathogenic index difference.NDV belongs to the member of the Rubulavirus (Rubulavirus) of Paramyxoviridae (Paramyxoviridae), paramyxovirus subfamily (Paramyxovirinae).Virus particle is polymorphism, and diameter is about 100～250nm, has the virus particle of cyst membrane generally rounded, but often irregular because of cyst membrane breakage form.(Newcastle disease virus is a non-segmented negative sub-thread minus-stranded rna virus NDV) to NDV, is the important member and the model virus of Paramyxoviridae.The full gene group leader of NDV 15186bp comprises 6 genes, respectively at least 8 kinds of albumen of coding; Be nucleoprotein (NP), phosphoric acid albumen (P), stromatin (M); Fusion rotein (F), hemagglutinin-neuraminidase (HN) and polymerase protein (L), and by V and two kinds of albumen of W of P genes encoding.Wherein F albumen is that the NDV cells infected is necessary, and it can not only promote virus envelope and host cell membrane to merge, and can promote to merge between the adjacent host cell, is the important component that constitutes the NDV virulence.

At present; The diagnosis of newcastle disease virulence mainly is serological test and egg inoculation experiment; Technical evaluation hypotypes such as real time fluorescence quantifying PCR method and gene chip have been developed in recent years successively; But existing pathogen separation and serological diagnostic method complex operation; Can't realize synchronous detection to different subtype, PCR method also can't realize to this virus carry out fast, accurately, the purpose of high throughput testing somatotype, thereby need that research is a kind of can carry out the integrated diagnostic techniques that hypotype was investigated, accurately differentiated to high-throughput fast to influenza virus.

Summary of the invention:

The objective of the invention is to overcome the shortcoming that prior art exists; Seek to provide the tetra-sodium sequencing technologies discrimination method of a kind of NDV virulent strain and low virulent strain, for bird aquaculture, meat product processing and aspects such as exporter and international meat-based food trade provide a kind of quick, easy, efficient, practical discrimination method.

To achieve these goals; Cardinal principle of the present invention is to utilize the tetra-sodium sequencing technologies; Through sequencing primer and the hybridization of PCR amplification single stranded deoxyribonucleic acid template; Hatch with deoxyribonucleic acid polymerase, adenosine triphyosphate (ATP) sulfurylase, luciferase, apyrase and substrate (APS), resorcinolphthalein, add four kinds of triphosphoric acid base deoxynucleotides (dNTPs) one by one: (triphosphoric acid adenyl-deoxyribonucleotide dATP; Triphosphoric acid thymidylic acid dTTP; Triphosphoric acid deoxycytidylic acid dCTP; Triphosphoric acid guanine deoxyribonucleoside acid dGTP), as matching with template, the end of this triphosphoric acid base deoxynucleotide and primer forms covalent linkage, discharges tetra-sodium group (PPi); Adenosine triphyosphate (ATP) sulfurylase catalysis tetra-sodium under the situation that substrate (APS) exists forms ATP; The resorcinolphthalein that ATP drives the luciferase mediation transforms to oxyluciferin, and oxyluciferin sends the visible light signal that is directly proportional with the adenosine triphyosphate amount; The optical signals charge-coupled device (CCD) is collected and is converted into the peak by software; The Nucleotide number that mixes is directly proportional in the peak height of each optical signal and the reaction; Adenosine triphyosphate and uncorporated triphosphoric acid base deoxynucleotide are degraded by apyrase; The cancellation optical signal; And regeneration reaction system, utilize optical signal to transform the peak figure that obtains, NDV virulent strain and low virulent strain in the sample are differentiated.

Design of primers is at first carried out in realization of the present invention, and the Auele Specific Primer and the tetra-sodium sequencing primer of NDV virulent strain and low virulent strain distinguished in design; Extract the Yeast Nucleic Acid (RNA) of testing sample again; Carry out rt-polymerase chain reaction (RT-PCR) amplification with Auele Specific Primer then; Carry out agarose gel electrophoresis again amplified production is detected,, then prepare tetra-sodium order-checking single-stranded template if the primer amplified fragment length is 268bp; Carry out the tetra-sodium sequencing reaction, judge in the sample whether contain NDV virulent strain or low virulent strain according to RT-PCR amplification and tetra-sodium sequencing result at last.

Design of primers of the present invention: according to the homology analysis result of DNASTAR software; Choose the sequence of NDV virulent strain and low virulent strain; Through Assay Design SW software design upstream primer, downstream primer and sequencing primer, primer sequence is respectively:

F upstream region of gene primer: 5 '-AAGCCCCCTTGGATGCATA-3 ', 5 ' carries out biotin labeling;

F gene downstream primer: 5 '-TCATGCACAGCCTCATTGG-3 ',

F gene sequencing primer: 5 '-CCAATAATGGCGCCT-3 ', expanding fragment length are 268bp.

The RNA of extraction testing sample of the present invention: be to utilize TRIZOL RNA extraction agent to handle testing sample, chloroform extracting protein, isopropanol precipitating obtains RNA; Or extract test kit with commercialization RNA and extract.

Of the present inventionly carry out RT-PCR reaction with Auele Specific Primer: the testing sample RNA that extracts is carried out reverse transcription, and reaction system comprises 3 μ L RNA templates, 2 μ L 25mmol/L magnesium chlorides (MgCl2); 1 μ L, 10 * RNAPCR damping fluid, 1 μ L2.5mmol/L triphosphoric acid base deoxynucleotide (dNTP), 0.25 μ L 40U/ μ L RNA enzyme inhibitors; 0.5 μ L 5U/ μ LAMV RNA ThermoScript II, 0.5 μ L 20pmol/ μ L F upstream region of gene primer and downstream primer is with diethylpyrocarbonate (DEPC; Diethypyrocarbonate) water is supplied volume to 10 μ L, instantaneous centrifugal mixing, and the reverse transcription condition is 42 ℃ of 30min; 98 ℃ of 1min, 4 ℃; Add 10 times of polymerase chain reaction damping fluids (10 * PCR buffer), 5 μ L, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L F upstream region of gene primer and downstream primer 0.5 μ L in 10 μ L cDNA (complementary DNA) solution after reverse transcription; DEPC water is supplied volume to 50 μ L; The PCR cycling condition is: behind 94 ℃ of preparatory sex change 5min, get into 94 ℃ of sex change 30s, 54 ℃ of annealing; 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then.

Preparation tetra-sodium order-checking single-stranded template of the present invention: use 50ul to be marked with the PCR product of vitamin H and the magnetic bead that the 200ug Streptavidin encapsulates, hatched at ambient temperature 20 minutes; To pick up with the PCR product after magnetic bead combines with vacuum prep tool, in 70% ethanol, clean 5s then; Sex change damping fluid (denatureation buffer) is washed 5s; Move on at last and clean 10s in the dcq buffer liquid (washing buffer); Vaccum prep tool puts into the plate that contains F gene sequencing primer, shakes, and discharges magnetic bead; Sample was put into 80 ℃ of baking ovens 2 minutes, again cool to room temperature.

The tetra-sodium sequencing reaction that carries out of the present invention: on PYROMARK ID instrument, detecting automatically under 28 ℃, application of sample uses 600 millibars/8 milliseconds application of sample pressure and time, the every wheel 65 seconds reaction times; Primer strand extends along with the adding of different triphosphoric acid base deoxynucleotides (dNTP); Along with the combination of nucleic acid, ccd video camera detects the optical signal that sends.

Of the present inventionly judge whether contain newcastle disease virulent strain or low virulent strain in the sample: utilize F upstream region of gene primer and downstream primer to carry out the RT-PCR amplification according to RT-PCR amplification and tetra-sodium sequencing result; If the RT-PCR reaction does not have the amplified fragments of 268bp, then do not contain newcastle disease virulent strain or low virulent strain in the sample; If the RT-PCR reaction has the amplified fragments of 268bp; Utilize F gene sequencing primer to carry out the tetra-sodium sequencing analysis again; Sequencing result is if contain the characteristic basic aminoacids sequence " Arg-Arg-Gln-Lys/Arg-Arg-Phe " of virulent strain; Judge that then sample contains the newcastle disease virulent strain, sequencing result judges that then sample contains Newcastle disease attenuated strain if do not contain the characteristic basic aminoacids sequence " Arg-Arg-Gln-Lys/Arg-Arg-Phe " of virulent strain.

The present invention has absorbed the advantage of RT-PCR and dna sequencing technology two aspects; The mensuration of using one section very short distinguished sequence satisfies the needs to molecular diagnosis; Make that the qualification result of virulent strain or low virulent strain of newcastle disease is more reliable, method is faster, and the test identification result is accurate.

Embodiment:

Further set forth the present invention through specific embodiment below.

Embodiment 1: differentiate whether the wild bird in Xinjiang infects newcastle disease virulent strain or low virulent strain

1, design specific primers sequence

Land the U.S. state-run biology information technology center (NCBI) through Internet; The NDV virulent strain that query and search has been announced and the nucleotide sequence of low virulent strain; Do not comprise not clear base components series; The same time to homology is nearer is only chosen a strain with the strain in place, with Editseq in DNAStar 7.1 software packages and MegAlign software the F gene nucleotide series of selected sample is edited, one by one comparison; With Clustal W Method (software) default parameters selected F gene nucleotide series is carried out homology relatively, find the conserved sequence region of virulent strain; The Auele Specific Primer of F gene and the design of sequencing primer are all carried out through Assay Design SW software, and sequence is following:

F upstream region of gene primer: 5 '-AAGCCCCCTTGGATGCATA-3 ', 5 ' carries out biotin labeling;

F gene downstream primer: 5 '-TCATGCACAGCCTCATTGG-3 ',

F gene sequencing primer: 5 '-CCAATAATGGCGCCT-3 ', expanding fragment length are 268bp.

2, extract the RNA of testing sample

Utilize TRIZOL lysate method to extract the RNA of testing sample, concrete steps are to get the 1.5mL centrifuge tube of sterilization, add 600 μ L TRIZOL lysates, add bird sample 200mg to be measured; Add 200 μ L chloroforms again, grind, vibration mixing 30s on the vortex mixer is in 4 ℃ of centrifugal 15min of 12000r/min; Draw supernatant 500 μ L and transfer in the new 1.5mL centrifuge tube, add-20 ℃ of precooling 500 μ L Virahols again, behind the precipitation at room temperature 30min, in 4 ℃ of centrifugal 15min of 12000r/min; Carefully remove supernatant, add 600 μ L, 70% ethanol, put upside down washing, in 4 ℃ of centrifugal 10min of 12000r/min; Remove supernatant again, centrifuge tube is inverted on the thieving paper drying at room temperature; Add 10 μ L ultrapure waters at last, mixing gently, the RNA on the dissolving tube wall, the centrifugal 5s of 2000r/min promptly obtains the RNA of sample; The RNA that extracts must carry out pcr amplification in 2 hours; Must place refrigerator if need prolonged preservation.

3, carry out the RT-PCR amplification with the F gene of NDV strain primer

The testing sample RNA that extracts is carried out the reverse transcription of F gene of NDV strain, and reaction system comprises 3 μ L RNA templates, 2 μ L 25mmol/L magnesium chlorides (MgCl2); 1 μ L, 10 * RNA PCR damping fluid, 1 μ L 2.5mmol/LdNTP (triphosphoric acid base deoxynucleotide), 0.25 μ L 40U/ μ L RNA enzyme inhibitors; 0.5 μ L 5U/ μ LAMV RNA ThermoScript II, 0.5 μ L20pmol/ μ L F gene of NDV strain upstream primer and downstream primer is with DEPC (diethypyrocarbonate; Diethylpyrocarbonate) water is supplied volume to 10 μ L, instantaneous centrifugal mixing, and the reverse transcription condition is 42 ℃ of 30min; 98 ℃ of 1min, 4 ℃.Add 10 * PCR buffer (10 times of polymerase chain reaction damping fluids), 5 μ L in 10 μ L cDNA (complementary DNA) solution after reverse transcription, 5U/ μ L Taq archaeal dna polymerase 0.25 μ L, 20pmol/ μ L F gene of NDV strain upstream primer and downstream primer 0.5 μ L; DEPC water is supplied volume to 50 μ L; The PCR cycling condition is: behind 94 ℃ of preparatory sex change 5min, get into 94 ℃ of sex change 30s, 54 ℃ of annealing; 72 ℃ are extended 30s, circulate altogether 50 times; 72 ℃ are extended 10min more then.

4, carry out agarose gel electrophoresis

The PCR product is carried out agarose gel electrophoresis, get the 2g agarose, in the 100mL electrophoretic buffer, heat, fully dissolve, adding the ethidium bromide stock solution is 0.5 μ g/mL to final concentration, glue.In electrophoresis chamber, add electrophoretic buffer, make liquid level just not have gel; 3 μ L～6 μ L pcr amplification products are mixed point sample with an amount of sample loading buffer respectively; 9V/cm constant voltage electrophoresis migrates to the gel middle part until the tetrabromophenol sulfonphthalein indicator; Ultraviolet Detector is observed electrophoresis result down, and sample to be checked can amplify specific band, and expanding fragment length is 268bp.

5, preparation tetra-sodium order-checking single-stranded template

Use 50 μ l to be marked with the PCR product of vitamin H and the magnetic bead that 200 μ g Streptavidins encapsulate, under 25 ℃ of room temperatures, hatched 20 minutes, will pick up with the PCR product after magnetic bead combines, in 70% ethanol, clean 5s then with vacuum prep tool; Denatureation buffer washes 5s; Move on at last among the washing buffer and clean 10s; Vaccum prep tool puts into the plate that contains F gene sequencing primer, shakes, and discharges magnetic bead; Sample was put into 80 ℃ of baking ovens 2 minutes, again cool to room temperature.

6, tetra-sodium order-checking

On tetra-sodium sequenator (PYROMARK ID), react, under 28 ℃ of conditions, application of sample uses 600 millibars/8 milliseconds application of sample pressure and time, the every wheel 65 seconds reaction times, and primer strand extends along with the adding of different dNTP; Along with the combination of nucleic acid, ccd video camera detects the optical signal that sends, and F gene tetra-sodium sequencing result is respectively ATAAAGGTAACGGGACCCC.

7, the result judges

The F gene amplification fragment length of sample to be checked is 268bp, and is consistent with known extension increasing sequence length; Through the tetra-sodium order-checking; The gene order of measuring is ATAAAGGTAACGGGACCCC; Through translation, this sequence does not contain the characteristic basic aminoacids sequence " Arg-Arg-Gln-Lys/Arg-Arg-Phe " of virulent strain, therefore judges that the wild bird in Xinjiang infects Newcastle disease attenuated strain.

Embodiment 2: whether the bird of differentiating Shandong plant infects newcastle disease virulent strain or low virulent strain

1, design specific primers sequence

With embodiment 1.

2, extract the RNA of testing sample:

The nasal cavity swab sample of getting the chicken crowd of plant is behind thorough mixing on the mixing tank; With the autoclaving tweezers liquid in the swab is extruded; Room temperature is placed 30min; Get supernatant, adopt RNA to extract test kit (TaKaRa MiniBEST Viral RNA/DNAExtraction Kit Ver 3.0), carry out the extraction of RNA to specifications.

3, carry out the RT-PCR amplification with the F gene of NDV strain primer

With embodiment 1.

4, carry out agarose gel electrophoresis

With embodiment 1, through observing, bird sample amplification to be checked goes out the specific band of 268bp.

5, preparation tetra-sodium order-checking single-stranded template

With embodiment 1.

6, tetra-sodium order-checking

With embodiment 1.

7, the result judges

The F gene amplification fragment length of sample to be checked is 268bp, and is consistent with known extension increasing sequence length; Through the tetra-sodium order-checking; The gene order of measuring is AGGAGACAAAAACGCTTT; Through translation, contain the characteristic basic aminoacids sequence " Arg-Arg-Gln-Lys/Arg-Arg-Phe " of virulent strain, judge that therefore the bird of Shandong plant infects the newcastle disease virulent strain.

The nucleotides sequence tabulation

< 110>Inspection and Quarantine Technology Center, Shandong Inspection and Quarantine

< 120>the tetra-sodium sequencing technologies is differentiated the method for NDV virulent strain and low virulent strain

<160>3

<170>PatentIn?version?3.5

<210>1

<211>19

<212>DNA

< 213>artificial sequence

<220>

<221>primer_bind

<222>(1)..(19)

< 223>be used for the upstream primer of NDV amplification F gene

<400>1

aagccccctt?ggatgcata 19

<210>2

<211>19

<212>DNA

< 213>artificial sequence

<220>

<221>primer_bind

<222>(1)..(19)

< 223>be used for the downstream primer of NDV amplification F gene

<400>2

tcatgcacag?cctcattgg 19

<210>3

<211>15

<212>DNA

< 213>artificial sequence

<220>

<221>primer_bind

<222>(1)..(15)

< 223>be used for the sequencing primer of NDV amplification F gene

<400>3

ccaataatgg?cgcct 15

Claims (1)

1. one kind is used for the primer that the tetra-sodium sequencing technologies is differentiated NDV virulent strain and low virulent strain, it is characterized in that:

F upstream region of gene primer: 5 '-AAGCCCCCTTGGATGCATA-3 ', 5 ' carries out biotin labeling;

F gene downstream primer: 5 '-TCATGCACAGCCTCATTGG-3 ',

F gene sequencing primer: 5 '-CCAATAATGGCGCCT-3 ', expanding fragment length are 268bp.