CN106282418A - A kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method - Google Patents

A kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method Download PDF

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CN106282418A
CN106282418A CN201610960353.5A CN201610960353A CN106282418A CN 106282418 A CN106282418 A CN 106282418A CN 201610960353 A CN201610960353 A CN 201610960353A CN 106282418 A CN106282418 A CN 106282418A
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csbv
bee
detection
primer
microsporidian
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黄少康
林丽花
黄伟峰
苏松坤
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Fujian Agriculture and Forestry University
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Abstract

The present invention is to provide a kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method, described primer is: CSBV 1 F:TATTCAGGGGGACGCTACAC, CSBV 1 R:GCGTGAGTTGACAGAAAATC；Nc F:GAGAGAACGGTTTTTTGTTTGAGA, Nc R:ATCCTTTCCTTCCTACACTGATTG.We establish highly sensitive duplex PCR detection method by primer, detect two kinds of cause of diseases by extracting midgut tissue RNA by PCR simultaneously, have reagent dosage few, and the advantage with high sensitivity and high accuracy.The method detects the sensitivity of CSBV and Nc simultaneously can distinguish as little as 6 and 3 copies.

Description

A kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method
Technical field
The invention belongs to honeybee diseases detection field, be specifically related to a kind of Chinese bee sacbrood and the micro-spore of Eastern bee Worm duplex PCR detection method.
Background technology
Chinese bee sacbrood and Eastern bee microsporidian are the important diseases of Eastern bee, respectively by (Chinese Sacbrood virus, CSBV) and and Eastern bee microsporidian (Nosema ceranae, Nc) cause, two kinds of cause of disease distributions Extensively, difficulty is cured, mainly to put prevention first.Therefore, the diagnosis fast and accurately of early disease is particularly important, i.e. be conducive to and Time take measures to carry out to block disease spread, can be again that seed selection, breeding work provide help.In the past, two kinds of Pathogen test were point Driving into capable, CSBV is as RNA viruses, it is necessary to first extracting RNA, after reverse transcription, then checks (two step method) with PCR, or directly uses RT-PCR method detection (one-step method).The infection detection most common method of Nc then, is extracted midgut tissue, is used microscope inspection Looking into and determine, this method detection sensitivity is low；After the DNA of the most useful extraction Nc, then with PCR detection.But have no detect simultaneously this two The method planting cause of disease.Therefore, we establish highly sensitive duplex PCR detection method, pass through by extracting midgut tissue RNA PCR detects two kinds of cause of diseases simultaneously, has reagent dosage few, and the advantage with high sensitivity and high accuracy.The method is simultaneously The sensitivity of detection CSBV and Nc can distinguish as little as 6 and 3 copies.
Summary of the invention
It is an object of the invention to provide a kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection side Method, detects two kinds of cause of diseases by extracting midgut tissue RNA by PCR simultaneously, has reagent dosage few, and have high sensitivity and The advantage of high accuracy.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection primer, honeybee metacercoid in described detection Sick primer is: CSBV-1-F:TATTCAGGGGGACGCTACAC, CSBV-1-R:GCGTGAGTTGACAGAAAATC；Detection east The primer Nc-F:GAGAGAACGGTTTTTTGTTTGAGA, Nc-R:ATCCTTTCCTTCCTACACTGATTG of Nosema apis worm.
A kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method, include the following:
(1) RNA of intestinal in sample is extracted；
(2) with One step RT-PCR, or first it is transcribed into cDNA, then the method for performing PCR expands；
(3) PCR reaction system 25 μ L: template 1 μ L, CSBV-1-F, CSBV-1-R primer described in 10 μm ol/L and detection east The each 1 μ L of primer, PCR Master Mix 12.5 μ L of Nosema apis worm；Residue H2O supplies；
(4) reaction condition: 94 DEG C of 3 min, 94 DEG C of 30 s, 60 DEG C of 30 s, 72 DEG C of 30 s, totally 35 circulations, 72 DEG C are prolonged again Stretch 5 min, after reaction terminates；
(5) 1% agarose gel electrophoresiies, gel-colored and observation；
(6) gel result interpretation: if there is 258bp characteristic bands, CSBV infects the positive, has the characteristic bands of 140bp, then east Side's Nosema apis insect infection is positive；If without corresponding band, then it is negative.
The application in preparation detection Chinese bee sacbrood and Eastern bee microsporidian reagent of the described primer.
It is an advantage of the current invention that:
SBV(Sacbrood virus) it is the cause of disease of Apis sac brood, cause the generation sac brood of Eastern bee CSBV is the one of SBV, and the middle honeybee of China infects universal, and it is serious to be injured.The detection of existing CSBV is also Molecular Detection means, I.e. realize by one-step method (RT-PCR) or two-step method (reverse transcription+PCR).In adult honeybee, the infection of Nosema apis worm is the most non- The most universal, the conventional detection method of Nc is microscopy, simple and quick, but precision is the lowest；Also there are the DNA extracting Nc, then the side of PCR detection Method.But at present, have no based on the duplex PCR method detecting both honeybee pathogens while RNA extraction.This method be based on The detection method that RNA extracts, has dramatically different with the detection method of the DNA extracting Nc.
Application this method, it is possible to achieve once extract, the advantage simultaneously carrying out two kinds of Pathogen test.And actually detected work In, for an Apis sample, common method is, or extracts the DNA of whole honeybee, or extracts the RNA of whole honeybee, only carries Take one.If simply extracting DNA, just cannot be carried out the detection of CSBV；If that extract is RNA, after reverse transcription synthesis cDNA, CSBV and Nc two to be used, to primer, carries out twice independent PCR reaction respectively, could realize the detection of two kinds of cause of diseases.And, What this method was extracted is midgut tissue, expends than the reagent extracted for whole honeybee and substantially reduces, and reaction times the most only needs once, And detection specificity and sensitivity are all suitable with single detection, therefore, have clear advantage.
Accompanying drawing explanation
The specificity of Fig. 1 duplex PCR method detection CSBV and Nc.M DNA molecular standard；1 CSBV is right without template blank According to；2 Nc are without template blank；3 (CSBV+Nc) are without template blank；4 CSBV positive sample；5 Nc positive samples This；6 (CSBV+Nc) superinfection positive sample.
The sensitivity of Fig. 2 duplex PCR method detection CSBV and Nc.1 CSBV is without template blank (NTC)；2 Nc without Template blank；3 (CSBV+Nc) are without template blank；Gradient dilution sample (the plasmid concentration of 4 ~ 7 CSBV plasmids It is followed successively by 6.05 × 102、6.05×101、6.05×100、6.05×10-1Copy)+CSBV primer；8 ~ 11 Nc plasmid (plasmids Concentration is followed successively by 2.97 × 102、2.97×101、2.97×100、2.97×10-1Copy)+Nc primer；12 ~ 15 CSBV(plasmids Concentration is successively with swimming lane 4 to 7)+decoding for DTMF；16 ~ 19 Nc plasmids (plasmid concentration is successively with swimming lane 8-11)+decoding for DTMF；20~ 23 (CSBV+Nc) plasmid+decoding for DTMF, wherein the plasmid concentration of CSBV is followed successively by 6.05 × 102、6.05×101、6.05× 100、6.05×10-1Copy；The plasmid concentration of Nc is followed successively by 2.97 × 102、2.97×101、2.97×100、2.97×10-1Copy Shellfish.
CSBV and Nc duplex PCR detection in honeybee worker bee in Fig. 3.M—DNA Marker；1 CSBV is without template blank； 2 Nc are without template blank；3 CSBV+Nc are without template blank；4 CSBV positive sample；5 Nc positive sample: 6 ~ The sample taken at random in 15 bee colonies.
Detailed description of the invention
Embodiment 1
Method:
(1) design primer；
Table 1 primer sequence
Note:CSBV-1—Chinese Sacbrood Bee Virus genome (655 ~ 893 nt)；*NcEastern bee microsporidian 16S RRNA gene, primer is quoted from document: FRIES I, CHAUZAT M P, CHEN Y P, et al. Standard methods For Nosema research [J]. JournalApicultural Research, 2015,52 (1): 1 28.).
(2) the middle intestinal of sample is obtained；
(3) RNA of intestinal in extracting；
(4) RT-PCR(one-step method is used), or first it is transcribed into cDNA, then the method (two-step method) of performing PCR expands；
(5) PCR reaction system (25 μ L): template 1 μ L, 10 μm ol/L upstream region of gene primers and each 1 μ L of downstream primer, PCR Master Mix 12.5 μ L, H2O 7.5μL。
(6) reaction condition: 94 DEG C of 3 min, 94 DEG C of 30 s, 60 DEG C of 30 s, 72 DEG C of 30 s, totally 35 circulations, 72 DEG C Re-extend 5 min, after reaction terminates.
(7) 1% agarose gel electrophoresiies, gel-colored and observation.
(8) gel result interpretation: if 258bp characteristic bands occurs, CSBV infects the positive, has the characteristic bands of 140bp, Then Eastern bee microsporidian infects the positive；If without corresponding band, then it is negative.
Interpretation of result:
The high specific of this method.As seen from Figure 1, purpose band is high-visible, is two kinds of cause of diseases to be detected through sequence verification.
The high sensitivity of this method.This method detects the sensitivity of CSBV and Nc simultaneously can distinguish as little as 6 and 3 copies. In Fig. 2, when the copy number of CSBV and Nc is the most as little as 6 and 2 (the 22nd swimming lane), still can detect.Fig. 3 is in middle bee colony Randomly drawing sample has carried out detection test, except swimming lane 7, outside 8,10,14, swimming lane 6,9,11 ~ 13,15 equal showed different Double infection.
This method protection domain applies also for two re-detections extracted based on RNA: after i.e. extracting RNA, i.e. can be straight to RNA Tap into row RT-PCR detection, it is also possible to advanced reverse transcription becomes cDNA, then carries out PCR amplification with cDNA.
This method protection domain applies also for all SBV(sacbrood virus) virus of family (includes other Apis Metacercoid virus (Sacbrood Virus), SBV Thailand strain (SBV Thai strain), SBV Korea strain (SBV Korean strain) etc., and Nosema apis worm (includes that Eastern bee microsporidian Nosema ceranae, Apis mellifera are micro- Sporozoon Nosema apis) two re-detections.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with Modify, all should belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>a kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
tattcagggg gacgctacac 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
gcgtgagttg acagaaaatc 20
<210> 3
<211> 24
<212> DNA
<213>artificial sequence
<400> 3
gagagaacgg ttttttgttt gaga 24
<210> 4
<211> 24
<212> DNA
<213>artificial sequence
<400> 4
atcctttcct tcctacactg attg 24

Claims (4)

1. a Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection primer, it is characterised in that: honeybee in detection The primer of sac brood is: CSBV-1-F:TATTCAGGGGGACGCTACAC, CSBV-1-R: GCGTGAGTTGACAGAAAATC。
2. a Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method, it is characterised in that:
Described method includes the following:
(1) RNA of intestinal in Apis sample is extracted；
(2) with One step RT-PCR, or first it is transcribed into cDNA, then the method for performing PCR expands；
(3) PCR reaction system 25 μ L: template 1 μ L, CSBV-1-F, CSBV-1-R primer described in 10 μm ol/L claim 1 and The each 1 μ L of primer, PCR Master Mix 12.5 μ L of detection Eastern bee microsporidian；Residue H2O supplies；
(4) reaction condition: 94 DEG C of 3 min, 94 DEG C of 30 s, 60 DEG C of 30 s, 72 DEG C of 30 s, totally 35 circulations, 72 DEG C are prolonged again Stretch 5 min, after reaction terminates；
(5) 1% agarose gel electrophoresiies, gel-colored and observation；
(6) gel result interpretation: if there is 258bp characteristic bands, CSBV infects the positive, has the characteristic bands of 140bp, then east Side's Nosema apis insect infection is positive；If without corresponding band, then it is negative.
A kind of Chinese bee sacbrood the most according to claim 2 and Eastern bee microsporidian duplex PCR detection method, It is characterized in that: the primer of described detection Eastern bee microsporidian is Nc-F:GAGAGAACGGTTTTTTGTTTGAGA, Nc-R:ATCCTTTCCTTCCTACACTGATTG.
4. primer as claimed in claim 1 is in preparation detection Chinese bee sacbrood and Eastern bee microsporidian reagent Application.
CN201610960353.5A 2016-11-04 2016-11-04 A kind of Chinese bee sacbrood and Eastern bee microsporidian duplex PCR detection method Pending CN106282418A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105755158A (en) * 2016-05-12 2016-07-13 福建农林大学 LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105755158A (en) * 2016-05-12 2016-07-13 福建农林大学 LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MA MINGXIAO等: "Molecular and Biological Characterization of Chinese Sacbrood Virus LN Isolate", 《COMPARATIVE AND FUNCTIONAL GENOMICS》 *
WEI-FONE HUANG等: "Comparative development and tissue tropism of Nosema apis and Nosema ceranae", 《JOURNAL OF INVERTEBRATE PATHOLOGY》 *
林丽花等: "二重PCR快速检测中蜂囊状幼虫病病毒与东方蜜蜂微孢子虫方法的建立", 《中国科技论文》 *

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Application publication date: 20170104