CN1229644C - Method for detecting nosema disease of bombyx mori and other insects and its kit - Google Patents
Method for detecting nosema disease of bombyx mori and other insects and its kit Download PDFInfo
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- CN1229644C CN1229644C CN 02125360 CN02125360A CN1229644C CN 1229644 C CN1229644 C CN 1229644C CN 02125360 CN02125360 CN 02125360 CN 02125360 A CN02125360 A CN 02125360A CN 1229644 C CN1229644 C CN 1229644C
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Abstract
The present invention relates to a method for diagnosing insect nosemosis (Nosema class) and a diagnosing reagent kit. The present invention has the technical scheme that a sample is processed by a DNA extracting solution prepared from an alkali and divalent metal ion chelating agent and an abstergent; DNA is extracted from the sample through phenol chloroform; a microparticle genome fragment is amplified by an attractor designed by a highly conservative DNA sequence in a microparticle (Nosema class) genome; whether insects are infected by the microparticles (Nosema class) can be detected by electrophoresis. The present invention has the advantages of low cost, convenient and simple use, strong specificity, high broad spectrum, etc., and can detect infection insects at an early pathogenesis stage.
Description
Technical field
The present invention relates to the method and the kit of a kind of diagnosis silkworm and other insect granulosis (Nosema genus).More specifically, the present invention relates to a kind ofly whether exist particulate 16S small subunit rRNA gene to detect the method and the diagnostic kit thereof of insect granulosis by detecting in the insect sample.
Technical background
The cause of disease of pebrine disease (Nosema bombycis) belongs to protozoan, in silkworm, infect popular through eating down with semina, has the susceptible early stage detection that is difficult for, be difficult for after ill preventing and kill off, characteristics such as propagation is fast, is difficult for eradicating, and is popular on a large scale easily, be the most serious crushing infectious disease on silkworm and mulberry are produced, classified as important quarantine and prevent and kill off object by each sericulture state.Particulate genus (Nosemasp.) microsporidian also is the important pathogen of many beneficial insects and insect in addition, as honeybee particulate, tussah particulate apiculture and tussah aquaculture are caused huge harm, and for example be used for the insect baculovirus and the parasitic wasp industry of biological control for extensive raising insect propagation, the harm that granulosis causes is also quite serious.On the other hand, the particulate that discharges some insect is again a kind of important biological control means, has play a part very positive as the atomic application of locust to the control of the plague of locusts.Thereby detect, diagnose the granulosis of insect effectively, and can play beneficial insect and stop pathogen infection, instruct the purpose of prevention, control in good time; Can play the evaluation prevention effect to insect, the effect that monitoring of diseases is popular.
Current pebrine disease detects, diagnoses the most that effective method is female moth microscopy method that Pasteur was set up in the eighties in 19th century, uses till today over more than 100 year always.This method is meant when the silkworm egg production of hybrid seeds, whether the female moth that produced ovum is ground the back has the particulate spore to exist in the microscopically Direct observation, to determine whether its silkworm seed of giving birth to infects granulosis, granulosis to other insect, whether the methods that adopt similar female moth detection method, promptly polypide to be detected being ground the back has atomic spore in the microscopically Direct observation more.Though female moth microscopy method or other microscopy method unusual practicability and effectiveness on producing, but be subjected to the limitation of method itself, there is considerable problem, influence detects effect, differ as reviewer's professional qualities, experience, can only the later stage detect in moth phase or morbidity, the sensitivity of detection is not high, inefficiency or the like.
The eighties in 20th century is along with development of biology, double immunodiffusion has appearred in succession, agglutination, fluorescence anti-body method, enzyme linked immunosorbent assay, the enzyme labelled antibody method, immunoperoxidase staining procedure, a series of immunological techniques such as monoclonal antibody method based on particulate surface antigen protein and antibody specificity reaction, be applied in the detection diagnosis of pebrine disease, obtained certain progress, but because these method cross reaction height, technical sophistication, particulate to not of the same race or subspecies will prepare different antibody, and the application cost height, and the cycle is long, reviewer's experimental skill is required high reason, so do not obtain large-scale promotion application so far.
PCR (PCR) is to utilize a pair of specific primer, with millions of times of ground amplifications of one section quick specificity of sequence in the dna profiling of denier, dna profiling molecule can reach the requirement of various detection meanss by pcr amplification in theory, is a kind of desirable means of early detection diagnosis granulosis.Have some at present and utilize round pcr to detect the trial of diagnosis granulosis, but also have suitable distance from practical application, main cause is because template DNA prepares very complicated, the cost height; Sensing range is narrow, majority can only detect a strain or a few strain particulate (Nosemabombycis), and ignored in a large number can infected insect other Nosema belong to atomic existence and detection; Do not carry out early stage (ovum phase) diagnosis of granulosis etc.PCR diagnostic techniques (Chen Xiu etc., 1996, silkworm industry science, 22 (4): 229-234 as existing pebrine disease; Caizhong is flat, 1997, silkworm industry science, 23 (4): 207-210), after the silkworm that will live adds liquid nitrogen homogenate, add protease K digesting, phenol: the chloroform extracting prepares the DNA of pcr template, the specific primer that utilizes them to design carries out the PCR reaction to template DNA, with agarose gel electrophoresis the PCR product is detected.But its PCR design of primers of this particulate detection technique only can detect 1-2 kind particulate only at nosema bombycis, does not relate to the particulate of other insect, does not attempt the early detection of granulosis.This method needs protease K digesting in application, steps such as liquid nitrogen homogenate make experimental cost very high, and it is centrifugal that the preparation of dna profiling also relates to multistep, processes such as phase transfer make loaded down with trivial details, the consuming time length of this method, therefore the PCR diagnostic techniques practicality of existing this granulosis is not strong, has no commercial value.
Summary of the invention
The object of the present invention is to provide the PCR diagnostic method of insect granulosis a kind of applied widely, easy to use, quick, cheap (Nosema genus), quarantine, diseases prevention etc. are checked, are allocated in the growth that is used for insect.
Another object of the present invention provides a kind of diagnostic kit that is used for insect granulosis (Nosema genus).
Insect granulosis PCR diagnostic method provided by the invention comprises the steps:
(a) preparation insect sample P CR reaction template to be measured: the insect sample is put into DNA extraction liquid, smashs mixing to pieces, adds the DNA extract, and is centrifugal, gets the supernatant dna profiling; DNA extraction liquid comprises the bivalent metal ion sequestrant of 1-100mM and the scaling agent of 0.1%-5% (W/V), and with alkaline matter the pH value of extract is transferred to 11-14;
(b) template in the Auele Specific Primer of detection 16S small subunit rRNA gene and the step (a) is carried out pcr amplification under the specific amplification condition;
(C) amplified production to step (b) carries out electrophoresis detection, and the existence of judging 16S small subunit rRNA gene according to electrophoresis result whether.
The wherein preferred NaOH of alkaline matter, KOH, the Na of DNA extraction liquid in (a)
2CO
3Or K
2CO
3Solution, more preferably NaOH solution.The preferred phenol chloroformic solution of DNA extract.Preferred EDTA of bivalent metal ion sequestrant or EGTA, more preferably EDTA, preferred concentration is the bivalent metal ion sequestrant of 5-20mM, the more preferably EDTA of 10mmol/L.The preferred SDS of scaling agent, Triton X-100 or Tween 80, more preferably SDS, preferred concentration is the scaling agent of 0.5-2% (W/V), more preferably 1% SDS.(b) Auele Specific Primer of used 16S small subunit rRNA gene is preferably the A primer in, i.e. primer 1:5 ' GTT GAT TCT GCC TGA GGT AGA CGC T3 ', primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 '; Or the B primer, i.e. primer 1 ': 5 ' CCT GTATGA TGA TCG ATG CAG3 ', primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 '.
The agarose gel electrophoresis of the preferred 0.5%-2% concentration of the electrophoresis (c).
In another aspect of the present invention, a kind of kit that detects the insect granulosis is provided, this kit comprises:
(a) preparation dna profiling required extract reagent: comprise the bivalent metal ion sequestrant of 1-100mM and the scaling agent of 0.1-5% (W/V), and the pH value of extract is transferred to 11-14 with alkaline matter;
(b) Auele Specific Primer and the required reagent of pcr amplification of detection 16S small subunit rRNA gene;
The wherein preferred NaOH of alkaline matter, KOH, the Na of DNA extraction liquid in (a)
2CO
3Or K
2CO
3Solution, more preferably NaOH solution.Preferred EDTA of bivalent metal ion sequestrant or EGTA, more preferably EDTA, preferred concentration is the bivalent metal ion sequestrant of 5-20mM, the more preferably EDTA of 10mmol/L.The preferred SDS of scaling agent, Triton X-100 or Tween 80, more preferably SDS, preferred concentration is the scaling agent of 0.5-2% (W/V), more preferably the SDS of 1% (W/V).(b) Auele Specific Primer of used 16S small subunit rRNA gene is preferably the A primer in, i.e. primer 1:5 ' GTT GAT TCTGCC TGA GGT AGA CGC T3 ', primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 '; Or the B primer, i.e. primer 1 ': 5 ' CCT GTA TGA TGA TCG ATG CAG3 ', primer 2: 5 ' CAATGG TAT CTA ATC ACC TTC G 3 '.Further add the mineral oil that comprises sterilization in the required reagent of pcr amplification in the preferred reagent box.
Pcr amplification condition of the present invention is: 92 ℃-97 ℃ (preferred 94 ℃) initial sex change earlier 5 minutes; Then with 92-97 ℃ (preferred 94 ℃) 30 seconds, 52-57 ℃ (preferred 55 ℃) 30 seconds, 71-73 ℃ (preferred 72 ℃) 30 seconds carry out 28-35 (preferred 30) circulation; Last 71-73 ℃ (preferred 72 ℃) extended 5-10 minute.
" reagent that pcr amplification is required " is meant that the gene with the denier in the template carries out the used reagent of pcr amplification reaction process, comprises dNTP, high temperature-resisting DNA polymerase, 10 * enzyme buffer liquid and distilled water etc.
Used sample can be any tissue, body fluid or the blood etc. that contain insect genes to be measured, and normally used is whole polypide.
The present invention is main laboratory facilities with round pcr, and according to the dna sequence dna of high conservative in the 16S small subunit rRNA gene order in particulate (Nosema genus) genome, design detects the Auele Specific Primer of 16S small subunit rRNA gene.
16S small subunit rRNA gene (Nosema sp.) sequence is:
caccaggttg?attctgcctg?acgtagacgc?tattccctaa?gattaaccca?tgcatgtttt 60
tgatatggaa?aaatggactg?ctcagtaata?ctcactttat?ttaatgtatt?aaattagtat 120
aactgcgtta?aagtgtagca?taagacatat?acagtaagag?tgagacctat?cagctagttg 180
ttaaggtaat?ggcttaacaa?ggcaatgacg?ggtgacggta?ttactttgta?atattccgga 240
gaaggagcct?gagagacggc?tactaagtct?aaggattgca?gcaggggcga?aacttgacct 300
atggatttta?tctgaggcag?ttatgggaag?taatattcta?ttgtttcata?ttgtaaaagt 360
atatgagatg?attaattgga?gggcaaatca?agtgccagca?gccgcggtaa?tacttgttcc 420
aagagtgtgt?atgatgattg?atgcagttaa?aaagtccgta?gtttatattt?aagaagcaat 480
atgaggtgta?ctgtatagtt?gggagagaga?tgaaatgtga?cgaccctgac?tggacgaaca 540
gaagccaaag?ctgtacactt?gtatgtattt?tttgaacaag?gacgtaagct?ggaggagcga 600
agatgattag?ataccattgt?agtccacagt?aaactatgcc?cgacgatgtg?atatgatatt 660
aattgtatta?gatgatagaa?atttgagttt?tttggctctg?ggatagtatg?atcgcaagat 720
tgaaaattaa?agaaattgac?ggaagaatac?cacaaggagt?ggattgtgcg?gcttaatttg 780
actcaacgcg?aggtaactta?ccaatatttt?attattcaga?gaagattttc?aatctgagaa 840
tgataatagt?ggtgcatggc?cgttttcaat?ggatgctgtg?aagttttgat?taatttcaac 900
aagacgtgag?acccttttat?taatagacag?acacaatcag?tgtaggaagg?aaaggattaa 960
aacaggtccg?ttatgccctc?agacattttg?ggctgcacgc?gcaatacaat?agatatataa 1020
tctttatggg?ataatatttt?gtaagagata?tttgaacttg?gaattgctag?taaattttat 1080
taaataagta?gaattgaatg?tgtccctgtt?ctttgtacac?accgcccgtc?gctatctaag 1140
atgatatgtg?ttgtgaaatt?agtgaaaact?acttgaacaa?tatgtattag?atctgatata 1200
agtcgtaaca?tggttgctgt?tggagaacca?ttagcaggat?cataa 1245
The Auele Specific Primer of 16S small subunit rRNA gene can be designed various primers with conventional method according to the coded sequence of known 16S small subunit rRNA gene, and designing this class primer is that those skilled in the art can be unlabored.The A primer design Nosema that will increase belongs to the sequence of 613bp between 619 bases of the 7th base to the in the 16S small subunit rRNA Gene Partial sequence; To the increase sequence of 192bp between the 428th base to 619 base of above-mentioned sequence of B primer design.These primers can increase and discern any one atomic this segment DNA sequence in the Nosema genus, and the biology that other non-Nosema belongs to is not reacted, therefore efficient, the high specificity (pointer belongs to effectively particulate Nosema) of the primer of designing by the method, broad spectrum activity height, can detect the various insects granulosis, as pebrine disease, the three-spotted plusia granulosis, mythimna separata granulosis, bollworm granulosis etc.
In order to carry out pcr amplification, it has been generally acknowledged that and to digest sample with Proteinase K that the albumen with removal and DNA combination discharges DNA.The present invention need not protease K digesting in the preparation of template, by digestive juice smudge cells and the atomic spore that alkali, sequestrant and scaling agent are formed, and released dna, and in experimental design, further simplified the step of existing extraction dna profiling; Not only reduced extraction cost, and easier to be quick.
Take commercialization to handle detection method of the present invention, make kit, kit comprises the required extract reagent of preparation dna profiling, the reagent that the PCR reaction is required; Can react the mineral oil that drips a small amount of sterilization in the required reagent at PCR, can freeze preservation in refrigerator and cooled, the term of validity can reach more than 1 year.The method of operating of by specification joins sample in the extract reagent, smashs the phenol chloroform extract that adds the same volume of autogamy behind the mixing to pieces, centrifugal 5 minutes of 10000rpm; The supernatant that takes a morsel joins in the PCR reaction reagent, carries out pcr amplification; Amplified production carries out electrophoresis detection can identify in the sample whether belonged to the particulate infection by Nosema.When detection of particles is sick, have only an application of sample step that adds the template DNA sample, simplified the application of sample operation greatly, shortened the application of sample time, reduced the chance of cross pollution.
Method of the present invention and kit all can make things convenient for and identify Nosema accurately and belong to particulate, compare with the PCR diagnostic techniques of the used granulosis in current experiments chamber to have following advantage:
Template prepare easy, cost low (be about existing diagnostic techniques cost 10%), the reaction time of expending few (the template extraction time of the present invention is about 1 hour, existing diagnostic method extraction time be about 6 hours).
2. high specificity (only at particulate Nosema belong to effectively), broad spectrum activity height (can detect the various insects granulosis), and can detect the early stage infected insect of morbidity.
Description of drawings
Fig. 1 detects silkworm and infects the particulate experiment
Wherein the A. template is the DNA of nosema bombycis for the silkworm B. template that infects granulosis,
C. template is healthy silkworm D.DNA molecular weight standard
Fig. 2 detects bollworm and infects the particulate experiment
Wherein the A. template is the atomic DNA of bollworm for the bollworm B. template that infects granulosis
C. template is healthy bollworm
Fig. 3 different content nosema bombycis DNA is through PCR kit testing result
Fig. 4 watches the testing result of feeding particulate silkworm different time
Fig. 5 different insects granulosis is through PCR kit testing result
The healthy silkworm DNA of A. nosema bombycis DNA B. wherein
C. the atomic silkworm D. of infected silkworm infects the atomic beet of beet armyworm night
Moth
D. infect the atomic mythimna separata F. of mythimna separata infect mythimna separata atomic in red lateral sulcus cocoon
Honeybee
Embodiment
Embodiment 1 this method detects silkworm and infects the granulosis experiment
(1) extraction of dna profiling
Get a silkworm that infects granulosis and put into the 0.5mL centrifuge tube, add 100 μ L DNA extraction liquid and (contain 10mmol/L EDTA, 1% (W/V) SDS, NaOH transfers solution to cause pH=13), smash sample to pieces with toothpick, place half an hour under the room temperature, add isopyknic phenol chloroform extract (phenol: chloroform=1: 1), suitably shake, centrifugal 5 minutes of 10000rpm, supernatant is a dna profiling.Positive control be with conventional method (Chen Xiu etc., 1996, silkworm industry science, 22 (4): 229-234) the nosema bombycis DNA of Ti Quing is a template; Negative control is the silkworm of a health, extracts dna profiling with above-mentioned dna profiling extracting method of the present invention.
(2) PCR reaction
The PCR reaction system is as follows:
Dna profiling 1 μ L
10 * Taq enzyme buffer liquid, 2.5 μ L
dNTP(10mM) 1μL
Primer 1 (10pM) 1 μ L
Primer 2 (10pM) 1 μ L
Taq enzyme (2U) 0.5 μ L
Distilled water 20.8 μ L
Taq enzyme buffer liquid is: 50mM Tris-HCl, 1mM DTT, 0.1mM EDTA, 100mM KCl, 1%Triton X-100,50% glycerine, pH8.0.Used primer is A primer (primer 1:5 ' GTTGAT TCT GCC TGA GGT AGA CGC T3 ', and primer 2: 5 ' CAA TGG TAT CTA ATC ACCTTC G 3 ').
Carry out the PCR reaction with the PCR instrument, reaction conditions is:
94 ℃ 5 minutes
72 ℃ 10 minutes
(3) detection of PCR reaction product
Get 5 μ L PCR reaction product, with Ago-Gel (electrophoresis 20 minutes is decided on concrete conditions such as electrophoresis tank, electrophoresis apparatuses for gel strength 0.8%, 40 volts of voltages) electrophoresis detection.
Electrophoresis result as shown in Figure 1, the electrophoretic band of dna molecular amount standard is represented 300bp respectively, 400bp, 500bp, 600bp, the dna fragmentation of 700bp and 800bp length, the silkworm (A) and the positive control (B) that infect granulosis have positive band near 600bp, healthy silkworm does not then have (C), illustrates that method of the present invention can effectively detect the granulosis of silkworm.
Embodiment 2 this method detect bollworm and infect the granulosis experiment
(1) extraction of dna profiling
Method is substantially with embodiment 1, and just sample is for infecting the bollworm of granulosis, and extract is: 2% Tween 80 and the EGTA of 10mmol/L, pH=11 (KOH accent).
(2) PCR reaction
Method is with embodiment 1, and just primer changes B primer (primer 1 ': 5 ' CCT GTA TGA TGA TCG ATG CAG3 ', primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 ') into.
(3) electrophoresis detection of PCR reaction product
Method is with embodiment 1.
This method can detect effectively and infect atomic bollworm.Electrophoresis result such as Fig. 2.The bollworm that infects granulosis has tangible electrophoretic band, and healthy bollworm does not then have electrophoresis band.
3 sub-sensitivity experiments of kit detection of particles of embodiment
The extracting method of conventional dna profiling is adopted in this experiment, and the DNA that extracts is quantitative, with PCR method DNA amplification of the present invention, the sensitivity of the PCR method that electrophoresis detection the present invention is used.
(1) extraction of dna profiling
The conventional same document of dna profiling extracting method (Chen Xiu etc., 1996, silkworm industry science, 22 (4): 229-234).
After the silkworm sample of will living adds liquid nitrogen homogenate, add Proteinase K liquid (Proteinase K 50 μ g/ml, 0.01M Tris, pH7.8,0.005M EDTA, 0.5% (W/V) SDS), 50 ℃ digested 3 hours.In sample, add isopyknic phenol: chloroform: isoamylol (25: 24: 1), content makes it to become emulsion in the mixing pipe, under the room temperature centrifugal 15 seconds (12,000g), water is transferred in another centrifuge tube, add isopyknic chloroform, mixing, under the room temperature centrifugal 15 seconds (12,000g), water is transferred in another centrifuge tube, the sodium acetate of the 3M of adding 1/10th, abundant mixing, the cold ethanol of 2 times of volumes of adding, low temperature is following 30 minutes behind the mixing, 0 ℃ down 12, centrifugal 10 minutes of 000g adds half 70% ethanol of pipe capacity, after the cleaning, the centrifugal ethanol of removing at room temperature, allows evaporate in the centrifuge tube, with an amount of TE dissolving DNA, obtain template DNA.
Nosema bombycis DNA behind gradient dilution for template (dna profiling content is respectively 200ng, 100ng, 50ng, 25ng, 12.5ng, 6.24ng, 3.13ng, 1.57ng)
Contrast CK: with healthy silkworm is that sample extracts DNA through above-mentioned conventional method, is 1160ng in order to the dna profiling content that carries out the PCR reaction.
(2) PCR reaction
Method is with embodiment 1.
(3) detection of PCR reaction product
Method is with embodiment 1.
The result:
The sensitivity of this kit detection can reach 1.57ng at least as can be seen from Figure 3.
The detection of the granulosis of different time behind embodiment 4 silkworm oral microparticles
Watch hello silkworm with nosema bombycis, took a sample after 1 day, be sampled to the 8th day day by day, as the used sample of present embodiment, in which day beginning can detect the granulosis silkworm to detect method of the present invention.
The silkworm of using the 1st day to the 8th day respectively is as the specimen preparation dna profiling, and template is used for the PCR reaction, the electrophoresis detection amplified production, and method is all with embodiment 1.
The result:
Watch hello particulate and can detect silkworm infection particulate after 3 days, the particulate that infect this moment does not also form spore in the silkworm body, therefore microscopically does not observe the particulate spore, that is to say, whether the silkworm sample that can not judge this moment with traditional microscopy method infects granulosis, illustrates that the present invention can carry out early detection to granulosis.
5 kits of embodiment are used the broad spectrum activity experiment
Sample:
A. nosema bombycis DNA
B. healthy silkworm DNA
C. the atomic silkworm of infected silkworm
D. infect the atomic beet armyworm of beet armyworm
E. infect the atomic mythimna separata of mythimna separata
F. infect the atomic Microplitis mediator (Haliday) of mythimna separata
Get six kinds of samples of above-mentioned A-F and prepare dna profiling with kit of the present invention, the performing PCR of going forward side by side reaction, amplified production carries out electrophoresis detection, and method is with embodiment 1.
The result:
As can be seen from Figure 5, infect Nosema not of the same race and belong to atomic silkworm, beet armyworm, mythimna separata, red lateral sulcus cocoon honeybee and can detect, illustrate that method of the present invention has broad spectrum activity in detecting the application that Nosema belongs to granulosis by this kit.
SEQUENCE?LISTING.txt
SEQUENCE?LISTING
＜110〉Tianyou Biologic Sci. ﹠ Tech. Co., Ltd., Chengdu
Institute of Zoology, Academia Sinica
＜120〉method and the kit of detection silkworm and other insect granulosis
<130>PI022108
<160>4
<170>Patent?In?version?3.1
<210>1
<211>1245
<212>DNA
<213>Nosema?sp.
<400>1
caccaggttg?attctgcctg?acgtagacgc?tattccctaa?gattaaccca?tgcatgtttt 60
tgatatggaa?aaatggactg?ctcagtaata?ctcactttat?ttaatgtatt?aaattagtat 120
aactgcgtta?aagtgtagca?taagacatat?acagtaagag?tgagacctat?cagctagttg 180
ttaaggtaat?ggcttaacaa?ggcaatgacg?ggtgacggta?ttactttgta?atattccgga 240
gaaggagcct?gagagacggc?tactaagtct?aaggattgca?gcaggggcga?aacttgacct 300
atggatttta?tctgaggcag?ttatgggaag?taatattcta?ttgtttcata?ttgtaaaagt 360
atatgagatg?attaattgga?gggcaaatca?agtgccagca?gccgcggtaa?tacttgttcc 420
aagagtgtgt?atgatgattg?atgcagttaa?aaagtccgta?gtttatattt?aagaagcaat 480
atgaggtgta?ctgtatagtt?gggagagaga?tgaaatgtga?cgaccctgac?tggacgaaca 540
gaagccaaag?ctgtacactt?gtatgtattt?tttgaacaag?gacgtaagct?ggaggagcga 600
agatgattag?ataccattgt?agtccacagt?aaactatgcc?cgacgatgtg?atatgatatt 660
aattgtatta?gatgatagaa?atttgagttt?tttggctctg?ggatagtatg?atcgcaagat 720
tgaaaattaa?agaaattgac?ggaagaatac?cacaaggagt?ggattgtgcg?gcttaatttg 780
actcaacgcg?aggtaactta?ccaatatttt?attattcaga?gaagattttc?aatctgagaa 840
tgataatagt?ggtgcatggc?cgttttcaat?ggatgctgtg?aagttttgat?taatttcaac 900
aagacgtgag?acccttttat?taatagacag?acacaatcag?tgtaggaagg?aaaggattaa 960
aacaggtccg?ttatgccctc?agacattttg?ggctgcacgc?gcaatacaat?agatatataa 1020
tetttatggg?ataatatttt?gtaagagata?tttgaacttg?gaattgctag?taaattttat 1080
taaataagta?gaattgaatg?tgtccctgtt?ctttgtacac?accgcccgtc?gctatctaag 1140
atgatatgtg?ttgtgaaatt?agtgaaaact?acttgaacaa?tatgtattag?atctgatata 1200
agtcgtaaca?tggttgctgt?tggagaacca?ttagcaggat?cataa 1245
<210>2
<211>25
<212>DNA
<213>Artificial
<220>
＜223〉primer
<400>2
gttgattctg?cctgaggtag?acgct 25
<210>3
SEQUENCE?LISTING.txt
<211>22
<212>DNA
<213>Artificial
<220>
＜223〉primer
<400>3
caatggtatc?taatcacctt?cg 22
<210>4
<211>21
<212>DNA
<213>Artificial
<220>
＜223〉primer
<400>4
cctgtatgat?gatcgatgca?g 21
Claims (11)
1. method that detects silkworm and other insect granulosis, it comprises step:
(a) preparation insect sample P CR reaction template to be measured: the insect sample is put into DNA extraction liquid, smashs mixing to pieces, adds the DNA extract, and is centrifugal, gets the supernatant dna profiling;
DNA extraction liquid comprises the bivalent metal ion sequestrant of 1-100mM and the scaling agent of 0.1%-5%W/V, and with alkaline matter the pH value of extract is transferred to 11-14;
(b) with template in Auele Specific Primer that detects 16S small subunit rRNA gene and the step (a), under the specific amplification condition, carry out pcr amplification;
(c) amplified production to step (b) carries out electrophoresis detection, and the existence of judging 16S small subunit rRNA gene according to electrophoresis result whether;
The 16S small subunit rRNA gene that described 16S small subunit rRNA gene is the insect genes group, its Auele Specific Primer is A primer or B primer;
Described A primer comprises primer 1:5 ' GTT GAT TCT GCC TGA GGT AGA CGC T3 ', and primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 ';
Described B primer comprises primer 1 ': 5 ' CCT GTA TGA TGA TCG ATG CAG3 ', and primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 '.
2. by the method for claim 1 described detection silkworm and other insect granulosis, it is characterized in that the alkaline matter in the DNA extraction liquid is NaOH, KOH, Na
2CO
3Or K
2CO
3Solution.
3. by the method for claim 1 described detection silkworm and other insect granulosis, it is characterized in that the DNA extract is the phenol chloroformic solution.
4. by the method for claim 1 described detection silkworm and other insect granulosis, it is characterized in that the bivalent metal ion sequestrant is EDTA or EGTA.
5. by the method for claim 1 described detection silkworm and other insect granulosis, it is characterized in that scaling agent is SDS, Triton X-100 or Tween 80.
6. by the method for claim 1 described detection silkworm and other insect granulosis, it is characterized in that electrophoresis is an electrophoresis in the Ago-Gel of 0.5%-2% concentration.
7. kit that detects silkworm and other insect granulosis, this kit comprises:
(a) extract reagent: contain the bivalent metal ion sequestrant of 1-100mM and the scaling agent of 0.1-5%W/V, and the pH value of extract is transferred to 11-14 with alkali; With
(b) Auele Specific Primer and the required reagent of pcr amplification of detection 16S small subunit rRNA gene;
The 16S small subunit rRNA gene that described 16S small subunit rRNA gene is the insect genes group, its Auele Specific Primer is A primer or B primer;
Described A primer comprises primer 1:5 ' GTT GAT TCT GCC TGA GGT AGA CGC T3 ', and primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 ';
Described B primer comprises primer 1 ': 5 ' CCT GTA TGA TGA TCG ATG CAG3 ', and primer 2: 5 ' CAA TGG TAT CTA ATC ACC TTC G 3 '.
8. by the kit of claim 7 described detection silkworm and other insect granulosis, it is characterized in that the alkaline matter in the extract reagent is NaOH, KOH, Na
2CO
3Or K
2CO
3Solution.
9. by the kit of claim 7 described detection silkworm and other insect granulosis, it is characterized in that the bivalent metal ion sequestrant in the extract reagent is EDTA or EGTA.
10. by the kit of claim 7 described detection silkworm and other insect granulosis, it is characterized in that the scaling agent in the extract reagent is SDS, Triton X-100 or Tween 80.
11. the kit by the described detection of claim 7 silkworm and other insect granulosis is characterized in that, adds the mineral oil of sterilization in the required reagent of pcr amplification.
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| CN 02125360 CN1229644C (en) | 2002-07-26 | 2002-07-26 | Method for detecting nosema disease of bombyx mori and other insects and its kit |
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| CN 02125360 CN1229644C (en) | 2002-07-26 | 2002-07-26 | Method for detecting nosema disease of bombyx mori and other insects and its kit |
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| CN1229644C true CN1229644C (en) | 2005-11-30 |
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