CN102154522B

CN102154522B - Primer and system for detecting bombyx mori cytoplasmic polyhedrosis virus (BmCPV)

Info

Publication number: CN102154522B
Application number: CN 201110085935
Authority: CN
Inventors: 刘吉平; 王永宾
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2011-04-07
Filing date: 2011-04-07
Publication date: 2013-01-30
Anticipated expiration: 2031-04-07
Also published as: CN102154522A

Abstract

The invention discloses a primer and method for detecting a bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The invention provides a pair of specific primers, i.e., BmCPVf and BmCPVr. A RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification program is designed, so that accurate and quick detection of the bombyx mori cytoplasmic polyhedrosis virus is realized. The method has high specificity, is quick and accurate for detection, is suitable for a small amount of sample operation, and is easy to operate. By adopting the method, the requirement on the molecular diagnosis of the BmCPV can be met, and technical bases are provided for early detection of production and prediction of midgut polyhedrosis of bombyx mori.

Description

One group of primer and detection system that is applied to detect the silkworm cytoplasmic polyhedrosis virus

Technical field

The invention belongs to molecular diagnosis and authenticate technology field, be specifically related to one group and be applied to detect the primer of silkworm cytoplasmic polyhedrosis virus and the method for rapid detection silkworm cytoplasmic polyhedrosis virus.

Background technology

The silkworm cytoplasmic polyhedrosis claims again mind-intesting cytoplasmic polyhedrosis, cause of disease be the silkworm cytoplasmic polyhedrosis virus ( Bombyx moriCytoplasmic polyhedrosis virus is called for short BmCPV), be the type species that Reoviridae matter polyhedrosis virus belongs to.Silkworm district, East China is in summer, autumn silkworm rearing season this disease that mostly occurs, then mostly occur at summer autumn silkworm rearing season high temperature season in silkworm district, south China, make the 3rd～6 of every year, investigating mind-intesting cytoplasmic polyhedrosis (being called for short again the BmCPV disease) according to us accounts for Guangdong the 3rd and makes silkworm 30～50% of the number of always falling ill, because this disease is normal and desonucleosis DNV disease is concurrent, be referred to as " spoken parts in traditional operas is young " disease in Guangdong.Sick silkworm mainly by family nibble lower pollution BmCPV mulberry leaf and in invading due to the intestines sequela.Sick silkworm after causing a disease grows slowly, and body body slight of stature is dull, and body colour is slightly milky white, and normal the discharge is with milky soft excrement, and is then dead successively.From infecting to falling ill about 6～10 days.Mind-intesting cytoplasmic polyhedrosis temperature relatively low 1,2,8 make the morbidity less; Each silkworm of high temperature make the morbidity more.In the Deqing County of 1986-1987 Guangdong Province Xijiang River bank, the flacherie census operations on the ground such as Wengyuan County of North Guangdong hilly and mountainous land finds that Deqing County Wang Wu village the 4th makes CPV sickness rate 8.12%, the village that has even up to 11.91%; Namely make with 3～7, daily mean temperature is 28～31 ℃, mostly occurs when sometimes reaching 34 ℃.

At present be divided into 9 strains according to its polyhedrosis shape and the position that in cell, forms, wherein the dsRNA gene complete sequence of BmCPV-I strain is determined, and the analysis of each gene order and the prediction of protein structure etc. have been reported, but also less (Lv Hongsheng, insect viruses molecular biology, Beijing are reported in the research to aspects such as its Gene Detecting BmCPV, Chinese agriculture science and technology press, 1998. 411～442), the technical blank that still belongs to is needed solution badly.

Summary of the invention

The objective of the invention is to overcome the deficiency of existing silkworm cytoplasmic polyhedrosis virus BmCPV detection technique, one group of primer that is applied to detect the silkworm cytoplasmic polyhedrosis virus is provided.

Another object of the present invention is based on described primer, to provide the method for a kind of rapid detection silkworm cytoplasmic polyhedrosis virus.Can effectively detect mind-intesting cytoplasmic polyhedrosis virus according to the inventive method, check that for producing upper early detection and precognition Midgut of Silkworm, Bombyx Mori type pus illness provides important technical foundation.

Purpose of the present invention is achieved by the following technical programs:

(AF 433660 to the present invention is based on the nucleotide sequence data of BmCPV dsRNA genome the 5th fragment of reporting on (http://www.ncbi.nlm.nih.gov/nuccore/AF433660.1) NCBI website, gi 1666064), design a pair of special primer, used the realization of RT-PCR technology to the detection of silkworm cytoplasmic polyhedrosis virus.

Described primer BmCPVf and BmCPVr, upstream primer 18bp is positioned at its 5' end from 2188bp to 2205bp, downstream primer 18bp, sequence between from 2400bp to 2417bp, the nucleotide sequence of two primers is respectively shown in SEQ ID NO:1 and SEQ ID NO:2:

BmCPVf：5' CAGAGCCATAATGAAACG 3' ；

BmCPVr：5' GTTGCTCCTGTTTTGTGG 3'；

The purpose fragment of amplification is 230 bp.

Simultaneously, the method for quick of silkworm cytoplasmic polyhedrosis virus provided by the invention may further comprise the steps:

(1) extracting silkworm cytoplasmic polyhedrosis virus genome dsRNA;

(2) the described primer of application carries out pcr amplification to the genome dsRNA of the silkworm cytoplasmic polyhedrosis virus BmCPV of extraction;

(3) get 5 μ L step (2) pcr amplification products with the agarose gel electrophoresis detection of 1.2 ﹪, carry out staining examine with nucleic acid dye Goldview.

25 μ L systems are adopted in the described PCR reaction of step (2): 2.5 μ L, 10 * PCR Buffer, and 1 μ L dNTPs, forward and reverse primer concentration is 10 μ M, respectively adds 1 μ L, 1 μ L dna profiling, 18 μ L ddH ₂O, 0.5 μ L TaqEnzyme (2.5 U/ μ L).The PCR circulation of primer amplification is: 94 ℃ of 5 min; 94 ℃ of 45 s, 50 ℃ of 45 s; 72 ℃ of 1 min, 30 circulations; 72 ℃ of 10 min, 4 ℃ of preservations.

The invention has the beneficial effects as follows:

The invention provides a pair of special primer BmCPVf/ BmCPVr, adopt described Auele Specific Primer effectively augmentation detection go out silkworm mind-intesting cytoplasmic polyhedrosis virus, the inventive method high specificity, detect quick and precisely, be suitable in a small amount sample operations, simple to operate, check that for producing upper early detection and precognition Midgut of Silkworm, Bombyx Mori type pus illness provides important technical foundation.

Description of drawings

The RT-PCR amplification of Fig. 1 silkworm cytoplasmic polyhedrosis virus genome dsRNA.

Embodiment

Further specify the present invention below in conjunction with the drawings and specific embodiments.Employed test method is ordinary method if no special instructions among the following embodiment; Employed material, reagent etc. if no special instructions, are reagent and the material that can obtain from commercial channels.

Embodiment 1

(1) artificial propagation of silkworm cytoplasmic polyhedrosis virus and purifying

Biomaterial: silkworm cytoplasmic polyhedrosis virus liquid is by the artificial mulberry leaf successive propagation of Agricultural University Of South China's silkworm and mulberry Biotechnology Experiment chamber warp (only use for explanation the inventive method, also can adopt the described silkworm cytoplasmic polyhedrosis virus of other laboratory related experiment uses); Agaroses (spanish) etc. are available from Guangzhou Bai Kang Bioisystech Co., Ltd; DL 2000 molecular weight standard nucleic acid are sky, Beijing root biochemical technology company limited product; Loading Buffer, TaqDNA Polymerase, dNTPs, DEPC are available from Beijing ancient cooking vessel state Bioisystech Co., Ltd; Chloroform, Virahol, dehydrated alcohol are produced by Tianjin Da Mao chemical reagent factory; TaKaRa PrimeScript ^TMRT-PCR Kit reverse transcription test kit is available from Rui Zhen company; The PCR instrument is PE Applied Biosystem 9700 produced in USA; Gel imaging system is the UVP that Britain produces.

Inoculation method: getting concentration is 10 ⁶The silkworm matter type polyhedron suspension of individual/mL is in 100 mL small beakers, mulberry leaf are cut into the fritter of 2cm * 2cm, in viral polyhedron suspension, soak (tow sides soak) with tweezers folder mulberry leaf, drop falls unnecessary cause of disease liquid on the small beaker wall of cup, then blade back be tiled in up four age molting silkworm on, add rear 6 hours of poison and give according to a conventional method mulberry with normal mulberry leaf, note observing the incidence of silkworm every day.

Collection method: the sick silkworm of picking from silkworm rearing bed, cut off its back with scissors, collect milky middle intestines in the triangular flask of sterilization, seal rear 4 ℃ of Refrigerator stores, to be purified.

Purification process: after intestines in the oyster white of collecting are added aqua sterilisa and grinds in mortar, be sub-packed in the centrifuge tube of 13 mL, the centrifugal 20min of 2500～3000 rpm abandons supernatant after balance, and precipitation adds water to 13 mL, mixing, and repeated centrifugation is washed 2～3 times after the balance.Abandon supernatant, visible precipitate is divided into three layers, and the upper strata is faint yellow, and the centre is oyster white, the bottom is black, add 2～3 mL aqua sterilisas, wipe gently the faint yellow precipitation in upper strata off with liquid-transfering gun first, sucking-off discards, add again 2～3 mL aqua sterilisas, milky white precipitate in the middle of the drawout carefully is collected in the 10mL centrifuge tube of sterilization 4 ℃ of Refrigerator stores gently.

（2）

(A) extracting of silkworm cytoplasmic polyhedrosis virus genome dsRNA

The extracting method of the genome dsRNA of the described infected silkworm cytoplasmic polyhedrosis virus of step (1) BmCPV is with reference to routine, specifically in the EP pipe, add the total RNA extraction agent of 1 ml Trizol(), add again 200 μ l silkworm matter type polyhedron suspension, rifle head pressure-vaccum mixing 1 min leaves standstill 5 min; Add 200 μ l chloroforms, violent jolting 15 ~ 30s leaves standstill 3 min; Centrifugal 5 min of 12000 rpm avoid rocking when whizzer takes out in the whizzer of 4 ℃ of precoolings, destroy water oil interface; The careful upper strata water of drawing changes in the new EP pipe, for avoiding being drawn onto intermediate interface, can draw less in right amount some waters; Add and the isopyknic Virahol of water (about 500 μ l), turn upside down 10 times, leave standstill 10 min; 4 ℃, centrifugal 10 min of 12000 rmp; Carefully remove supernatant, suck the residual liquid in the pipe end with the rifle head; 75% ethanol that adds-20 ℃ of precoolings rotates the EP pipe for several times gently, makes ethanol infiltrate precipitation and tube wall; 4 ℃, centrifugal 5 min of 7500 rpm; Abandon supernatant, suck the residual ethanol in the pipe end with the rifle head, complete to the ethanol volatilization about dry 5 min of spacious lid in the super clean bench; Add an amount of DEPC water 20 μ l dissolving RNA precipitation; Get 5 μ l electrophoretic examinationss, all the other packing are stored in-70 ℃, or directly do reverse transcription.

(B) extracting of normal domestic silkworm gene group RNA

The silkworm of liquid nitrogen cryopreservation is put in the mortar of Liquid nitrogen precooler and grinds to form powdery, then be sub-packed in the centrifuge tube of 1.5 mL.The method of the normal domestic silkworm gene group of extracting RNA is with above-mentioned extracting BmCPV geneome RNA.

(3) RT-PCR amplification

(AF 433660 according to the nucleotide sequence data of BmCPV dsRNA genome the 5th fragment of reporting on the NCBI website, gi 1666064) (http://www.ncbi.nlm.nih.gov/nuccore/AF433660.1), design suitable RT-pcr amplification primer BmCPVf/ BmCPVr, upstream primer 18bp, be positioned at its 5' end from 2188bp to 2205bp, downstream primer 18bp, the sequence between from 2400bp to 2417bp, the nucleotide sequence of two primers is respectively:

BmCPVf：5' CAGAGCCATAATGAAACG 3' ；

BmCPVr:5'GTTGCTCCTGTTTTGTGG 3'; The purpose fragment of amplification is 230 bp.

Above-mentioned primer is synthetic by the handsome Bioisystech Co., Ltd in Shanghai.The reverse transcription reaction of all geneome RNA templates uses TaKaRa PrimeScript TM RT-PCR Kit reverse transcription test kit, then reverse transcription product is carried out pcr amplification with above-mentioned primer, 25 μ L systems are adopted in the PCR reaction: 2.5 μ L, 10 * PCR Buffer, 1 μ L dNTPs, forward and reverse primer concentration is 10 μ M, respectively add 1 μ L, 1 μ L dna profiling, 18 μ L ddH ₂O, 0.5 μ L TaqEnzyme (2.5 U/ μ L).The PCR circulation of primer amplification is: 94 ℃ of 5 min; 94 ℃ of 45 s, 50 ℃ of 45 s; 72 ℃ of 1 min, 30 circulations; 72 ℃ of 10 min, 4 ℃ of preservations.Get 5 μ L PCR products with the agarose gel electrophoresis detection of 1.2 ﹪, carry out staining examine with nucleic acid dye Goldview.

Experimental result is seen shown in the accompanying drawing 1, uses the inventive method to the RT-PCR detected result of silkworm cytoplasmic polyhedrosis virus genome dsRNA, and a clearly band is arranged below 250bp, and is consistent with expected result.Swimming lane: M. DL2000 DNA molecular weight standard; 1. the RT-PCR product of BmCPV genome dsRNA; 2. the RT-PCR product of healthy domestic silkworm gene group RNA; 3. ddH ₂O.

Experiment showed, the Auele Specific Primer that the present invention designs, be applied to the RT-PCR augmentation detection of described virus, augmentation detection goes out mind-intesting cytoplasmic polyhedrosis effectively, checks that for producing upper early detection and precognition Midgut of Silkworm, Bombyx Mori type pus illness provides important experimental basis.

SEQUENCE LISTING

＜110〉Agricultural University Of South China

＜120〉one group of primer and detection method that is applied to detect the silkworm cytoplasmic polyhedrosis virus

<130>

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> DNA

＜213〉primer BmCPVf

<400> 1

cagagccata atgaaacg 18

<210> 2

<211> 18

<212> DNA

＜213〉primer BmCPVr

<400> 2

gttgctcctg ttttgtgg 18

Claims

1. one group of primer that is applied to detect the silkworm cytoplasmic polyhedrosis virus is characterized in that its nucleotide sequence is respectively shown in SEQ ID NO:1 and SEQ ID NO:2.

2. the application of the described primer of claim 1 is characterized in that Application and preparation is in the reagent of the context of detection of silkworm cytoplasmic polyhedrosis virus.

3. application rights requires 1 described primer the genome dsRNA of the silkworm cytoplasmic polyhedrosis virus BmCPV of extraction to be carried out the system of pcr amplification, it is characterized in that described pcr amplification adopts 25 μ L systems: 2.5 μ L, 10 * PCR Buffer, 1 μ L dNTPs, forward and reverse primer concentration is 10 μ M, respectively add 1 μ L, 1 μ L dna profiling, 18 μ L ddH ₂O, concentration is 2.5 U/ μ L's TaqEnzyme 0.5 μ L;

The nucleotide sequence of described forward and reverse primer is respectively shown in SEQ ID NO:1 and SEQ ID NO:2.