CN109439801A - A kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box and its detection method - Google Patents
A kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box and its detection method Download PDFInfo
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Abstract
The present invention relates to a kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box and its detection methods, are exclusively used in the detection of honeybee Israel acute paralysis virus.The kit includes: (1) RT-PCR reaction solution；(2) Taq archaeal dna polymerase；(3) reverse transcriptase；(4) positive criteria product；(5) negative control；(6) sterile water.The invention has the following advantages that (1) good stability and specificity: there is high degree of specificity to the detection of honeybee Israel acute paralysis virus, and other honeybee virus no cross reactions, and it is reproducible；(2) high sensitivity: sensitivity can reach 10 copies/μ L；(3) easy to operate, quick: entire reaction can be completed in 2 hours.The detection and its antidiastole with other honeybee virosis that kit of the present invention can be used for honeybee Israel acute paralysis virus, early prevention and treatment in time to the disease are of great significance.
Description
Technical field
The present invention relates to a kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box and its detections
Method belongs to animal health technical field, suitable for honeybee Israel acute paralysis virus detection and its with other honeybees disease
The antidiastole of viral disease.
Background technique
Israel's acute paralysis viral (Israeli acute paralysis virus, IAPV) is Israel in 2004
Ilan Sela of Hebrew University et al. has found that a kind of new virus, the virus are sense single stranded rna virus, and one without envelope
Icosahedron protein coat, belong to imitative picornavirus mesh (picorna-like virus) bicistronic mRNA Viraceae
(Dicistroviridae) member.IAPV can infect each developmental stage honeybee individual (ovum, larva, pupa, at bee) and bee colony
Type honeybees (worker bee, drone and queen bee) at different levels, illness honeybee body colour is dimmed, villus shedding, trembles with wing, and gradually paralysis is dead
It dies, will cause a large amount of losses of honeybee, bee raising industry is endangered big.IAPV is widely distributed, Europe, South America and Asia etc.
There are relevant report in multiple countries.
Currently, there is scientist to research and analyse, IAPV is likely to lead to bee colony collapse imbalance disease (Colony collapse
One of disorder, CCD) main pathogenic, a large amount of disappearances of honeybee can be caused, honeybee loss quantity can reach entire bee
The 80% ~ 100% of group's sum, so as to cause serious pollination crisis.China is bee-keeping big country, the world, quantity of raising bees and bee product
Yield is sure to occupy always first place in the world for many years.The stable development of apiculture for promote increasing peasant income, improve crop yield and
Maintaining ecological balance is all of great significance.But honeybee disease, which is frequently broken out to have become, influences China's apiculture health hair
The principal element of exhibition, wherein the harm of honeybee virus will especially cause to pay close attention to.Therefore it establishes one kind and quickly, accurately detects IAPV's
Method prevents the infection of IAPV most important in turn.
Immunology detection and nucleic acid molecules detection technique are mainly used to the detection of honeybee virosis at present.Immune detection is
The method of common quickly detection virus a kind of at present, but the genetic homology with height between many viruses of honeybee, are answered
It is poor to will cause honeybee viral diagnosis specificity with immunological technique, is difficult to carry out Accurate Diagnosis to honeybee virosis.Based on core
Compared with immunoassay technology, round pcr has obtained in terms of pathogen detection revolutionary the PCR detection technique of acid molecule
Success, become the goldstandard that nucleic acid quickly detects, have susceptibility height, high specificity, simplicity, quickly and accurately, be
One of the detection technique that most common and laboratory mainly uses.Real-time fluorescence RT-PCR method is by utilizing fluorescence
Probe further enhances specificity, directly displays amplification, without electrophoresis detection, more shortens detection time.
IAPV disease symptoms and bee acute paralysis virus (acute bee paralysis virus, ABPV) are similar, raw
Object and phylogenetic systematics characteristic and ABPV and honeybee Kashmir viral (Kashmir bee virus, KBV) connect very much
Closely, in addition, the homology of these three nucleic acid sequences of IAPV, ABPV and KBV is all very high, reach 65% or more.Pass through sequence ratio
It is found to analysis, the polymerase polyprotein gene relative to ABPV, KBV and IAPV has relatively high special
Property, and the gene is relatively conservative for IAPV, and the specificity of method can be improved.
The present invention utilizes the conservative of honeybee Israel acute paralysis virus polymerase polyprotein gene specific
Primers and probe are developed by the optimization to reaction system for detecting honeybee Israel acute paralysis virus
Real-time fluorescence RT-PCR kit and method, this method can complete the quick detection of IAPV in 2 h.Currently, detection honeybee with
The real-time fluorescence RT-PCR kit and its detection method of color column acute paralysis virus have not been reported, which makes up the current country
Technical need in relation to the detection of bee class epidemic disease, the sound development of bee colony and natural ecology in protectorate.
Summary of the invention
The purpose of the present invention is to provide a kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagents
Box and its detection method make up the deficiency of existing detection technique, have the characteristics that high specificity, high sensitivity, easy to operate,
Can honeybee Israel acute paralysis virus in honeybee and its bee product fast and accurately be quarantined and be identified.
In order to achieve the object of the present invention, technical scheme is as follows:
A kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box, it is characterised in that: draw including forward direction
Object IAPV-F, reverse primer IAPV-R and TaqMan probe IAPV-P, each primer nucleotide sequences are as follows:
Forward primer IAPV-F:5 '-ACTAGTGGACGAAGCGAGTT-3 ',
Reverse primer IAPV-R:5 '-TGTACTGGGCAGTTACAGCA-3 '；
TaqMan probe IAPV-P:5 '-CGGAACGCCGCATGTGTTACCA-3 ',
5 ' the ends and 3 ' ends of probe are respectively adopted fluorophor FAM and BHQ1 and are modified.
Wherein, the kit includes following reagent:
(1) RT-PCR reaction solution: the RT-PCR reaction solution constituent of 50 reaction systems are as follows: 5 × One Step RNA PCR
250 μ L of Buffer, concentration are each 25 μ L of forward primer IAPV-F and reverse primer IAPV-R of 10 μm of ol/L, and concentration is 5 μ
The 50 μ L of TaqMan probe IAPV-P of mol/L, concentration are the 25 μ L of dNTP of 10 mmol/L, and concentration is 25 mmol/L's
MgCl2100 μ L, concentration are the 25 μ L of RNase Inhibitor of 40 U/ μ L, amount to 500 μ L；- 20 DEG C of preservations；
(2) Taq DNA polymerase: 25 μ L concentration are 5 U/ μ L Taq DNA polymerase 1, -20 DEG C of preservations；
(3) reverse transcriptase: 25 μ L concentration are 5 U/ μ L reverse transcriptase 1, -20 DEG C of preservations；
(4) positive criteria product: 100 μ L concentration are 1 × 103Copy/μ L positive criteria product 1, using contain target DNA piece
The positive plasmid of section is as positive criteria product, -20 DEG C of preservations；
(5) negative control: 100 μ L concentration are negative control 1 of 100 ng/ μ L, using the total of healthy honeybee tissue extraction
RNA is negative control sample, -20 DEG C of preservations；
(6) sterile water: 2 mL sterile waters 2.
Above-mentioned honeybee Israel acute paralysis virus real-time fluorescence RT- is used another object of the present invention is to provide a kind of
The detection method of PCR detection kit, comprising the following steps:
(1) RT-PCR reaction system configures: it is poly- that RT-PCR reaction solution 10.0 μ L, 5 U/ μ L Taq DNA being added in PCR pipe
Then synthase 0.5 μ L, 5 U/ μ L reverse transcriptase 0.5 μ L, 2.0 μ L of sample to be tested RNA are added 12.0 μ L of aqua sterilisa, make
Reaction total volume is 25.0 μ L；
(2) RT-PCR response procedures: 42 DEG C of 15 min of reverse transcription；95 DEG C of 3 min of initial denaturation；Then 95 DEG C of denaturation 5 s, 61
DEG C annealing extend 30 s, totally 40 circulation, in 61 DEG C of progress single-point fluorescence detections；
(3) result judgement: negative control is without Ct value and without amplification curve, while value≤35 positive control Ct, and typical case occurs
Amplification curve, illustrate experiment for effectively experiment, otherwise experiment is invalid；In the case where testing effective situation, sample to be tested is without Ct value
And without amplification curve, indicate in sample without honeybee Israel acute paralysis virus；Value≤35 sample to be tested Ct, and occur typical
Amplification curve, indicate sample in containing honeybee Israel acute paralysis virus；Sample to be tested Ct value > 35, then to the sample weight
Reinspection is surveyed: repeating testing result, then the sample is viral without honeybee Israel acute paralysis without Ct value；Repeating testing result has Ct
It is worth in the then sample containing honeybee Israel acute paralysis virus.
The invention has the following advantages that (1) good stability and specificity: system's selection of the present invention honeybee Israel is acute
Paralysis virus polymerase polyprotein gene conservative fragments are target, not only devise a pair of of specific primer,
And a specificity fluorescent probe has also been devised, dual control can be carried out to target in fluorescence RT-PCR reaction process
System, to the detection of honeybee Israel acute paralysis virus with high degree of specificity, and other honeybee virus no cross reactions, and again
Renaturation is good；(2) high sensitivity: the present invention uses TaqMan-BHQ1 probe, by the optimization of reaction system and reaction condition, into
One step promotes its sensitivity, can reach 10 copies/μ L；(3) easy to operate, quick: real-time fluorescence collects data, does not need to carry out
The amplification situation of electrophoresis detection nucleic acid, entire reaction can be completed in 2 hours.
Detailed description of the invention
Fig. 1 is the positive criteria product and negative control RT-PCR amplification of embodiment 1.Wherein M:100bp Ladder
DNA Marker I；1: positive criteria product, size are about 127 bp；2: negative control.
Fig. 2 is the detection method of Israel's acute paralysis virus real-time fluorescent RT-PCR detection reagent box of embodiment 2
Annealing temperature optimum results.When annealing temperature is 61.4 DEG C, fluorescence intensity is most strong, so in the kit test method most
Good annealing temperature is 61 DEG C.
Fig. 3 is the kinetic curve of the honeybee Israel acute paralysis virus positive criteria product fluorescence RT-PCR of embodiment 4.
Abscissa represents the recurring number of fluorescence RT-PCR amplification in figure, and ordinate represents fluorescence signal intensity；Amplification curve from right to left
The concentration of positive criteria product is 1 × 10 respectively1To 1 × 108Copy/μ L.
Fig. 4 is that the standard of the honeybee Israel acute paralysis virus positive criteria product real-time fluorescence RT-PCR of embodiment 4 is bent
Line.Abscissa represents standard items copy numerical value in figure, and ordinate represents RT-PCR amplification cycles number；Calibration curve equation be Y=-
3.479x+39.841；R represents related coefficient, coefficient of determination R2It is 0.973, amplification efficiency 98.359%.
Fig. 5 is that the specificity of 5 honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box of embodiment is surveyed
Determine result.Wherein 1: honeybee Israel acute paralysis virus；2: black Beequeen table virus；3: honeybee metacercoid virus；4: acute
Paralysis virus；5: vestigial wing virus；6: negative control.
Fig. 6 is the clinical sample testing result of embodiment 6.Including the positive sample of positive control, negative control and 5 parts
Product.
Specific embodiment
In order to which the present invention is furture elucidated rather than the limitation present invention, it is illustrated with reference to embodiments.Following implementations
Experimental method described in example is unless otherwise specified conventional method；The reagent and biomaterial are unless otherwise specified
It obtains from commercial channels.
Embodiment 1:
A kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box, including it is forward primer IAPV-F, anti-
To primer I APV-R and TaqMan probe IAPV-P, wherein forward primer IAPV-F sequence is 5 '-
ACTAGTGGACGAAGCGAGTT -3 ', reverse primer IAPV-R sequence are 5 '-TGTACTGGGCAGTTACAGCA -3 '；
TaqMan probe IAPV-P sequence is 5 '-FAM- CGGAACGCCGCATGTGTTACCA-BHQ1-3 '.Under the kit includes
Column reagent (50 reaction systems):
(1) RT-PCR reaction solution: the RT-PCR reaction solution constituent of 50 reaction systems are as follows: 5 × One Step RNA PCR
250 μ L of Buffer, concentration are each 25 μ L of forward primer IAPV-F and reverse primer IAPV-R of 10 μm of ol/L, and concentration is 5 μ
The 50 μ L of TaqMan probe IAPV-P of mol/L, concentration are the 25 μ L of dNTP of 10 mmol/L, and concentration is 25 mmol/L's
100 μ L of MgCl2, concentration are the 25 μ L of RNase Inhibitor of 40 U/ μ L, amount to 500 μ L；- 20 DEG C of preservations；
(2) Taq DNA polymerase: 25 μ L concentration are 5 U/ μ L Taq DNA polymerase 1, -20 DEG C of preservations；
(3) reverse transcriptase: 25 μ L concentration are 5 U/ μ L reverse transcriptase 1, -20 DEG C of preservations；
(4) positive criteria product: 100 μ L concentration are 1 × 103Copy/μ L positive criteria product 1, using contain target DNA piece
The positive plasmid of section is as positive criteria product, -20 DEG C of preservations；
(5) negative control: 100 μ L concentration are negative control 1 of 100 ng/ μ L, using the total of healthy honeybee tissue extraction
RNA is negative control sample, -20 DEG C of preservations；
(6) sterile water: 2 mL sterile waters 2.
Embodiment 2:
The annealing temperature of the detection method of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box optimizes
(1) RT-PCR reaction solution 10.0 μ L, 5 U/ μ L Taq RT-PCR reaction system: are separately added into each PCR pipe
0.5 μ L of DNA polymerase, 5 U/ μ L reverse transcriptase, 0.5 μ L, 10 times are incremented by diluted 2.0 μ L of standard sample, are then added
12.0 μ L of aqua sterilisa makes to react 25.0 μ L of total volume；
(2) RT-PCR optimizes response procedures: 42 DEG C of 15 min of reverse transcription；95 DEG C of 3 min of initial denaturation；Then 95 DEG C of 5 s of denaturation,
55 DEG C -65 DEG C (gradient temperature) annealing extend 30 s, and 3 repetitions are arranged in each gradient temperature, totally 40 circulations；
(3) annealing temperature selects: annealing temperature optimum results are as shown in Fig. 2, amplified production fluorescence when annealing temperature is 61.4 DEG C
Intensity highest, therefore select 61 DEG C of annealing temperatures as the kit response procedures.
Embodiment 3:
Use the detection method of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box, including following step
It is rapid:
(1) RT-PCR reaction system configures: it is poly- that RT-PCR reaction solution 10.0 μ L, 5 U/ μ L Taq DNA being added in PCR pipe
Then synthase 0.5 μ L, 5 U/ μ L reverse transcriptase 0.5 μ L, 2.0 μ L of sample to be tested RNA are added 12.0 μ L of aqua sterilisa, make
Reaction total volume is 25.0 μ L；
(2) RT-PCR response procedures: 42 DEG C of 15 min of reverse transcription；95 DEG C of 3 min of initial denaturation；Then 95 DEG C of denaturation 5 s, 61
DEG C annealing extend 30 s, totally 40 circulation, in 61 DEG C of progress single-point fluorescence detections；
(3) result judgement: negative control is without Ct value and without amplification curve, while value≤35 positive control Ct, and typical case occurs
Amplification curve, illustrate experiment for effectively experiment, otherwise experiment is invalid；In the case where testing effective situation, sample to be tested is without Ct value
And without amplification curve, indicate in sample without honeybee Israel acute paralysis virus；Value≤35 sample to be tested Ct, and occur typical
Amplification curve, indicate sample in containing honeybee Israel acute paralysis virus；Sample to be tested Ct value > 35, then to the sample weight
Reinspection is surveyed: repeating testing result, then the sample is viral without honeybee Israel acute paralysis without Ct value；Repeating testing result has Ct
It is worth in the then sample containing honeybee Israel acute paralysis virus.
Embodiment 4:
The foundation of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box examination criteria curve:
(1) standard items DNA dilutes: 10 times of positive colony plasmid sterile water containing target fragment being incremented by and are diluted to 1 × 108
Copy/L ~ 1 × 10 μ1A series of Plasmid DNA standard items of copy/μ L；
(2) calibration curve equation is established: using the Plasmid DNA standard items after diluting as template, each standard sample processing repeats 3
It is secondary, and be control with healthy honeybee total tissue RNA, using sterile water as blank control.25 μ L are configured by mode described in embodiment 3
Real-time fluorescence RT-PCR reaction system and carry out real-time fluorescence RT-PCR reaction；Amplification curve as shown in Figure 3 is obtained, then
Standard curve as shown in Figure 4 is drawn, calibration curve equation is constructed.The present embodiment building calibration curve equation be Y=-
3.479x+39.841；R represents related coefficient, coefficient of determination R2It is 0.973, amplification efficiency 98.359%.
Embodiment 5: the specific assay of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box
Viral with honeybee Israel acute paralysis respectively, black Beequeen table is viral, honeybee metacercoid is viral, acute paralysis is viral,
5 kinds of vestigial wing virus etc. common honeybee viruses are sample, carry out RT- after extraction RNA according to a conventional method in the method for embodiment 3
PCR reaction and result judgement, as shown in Figure 5, only honeybee Israel acute paralysis virus, which has, generates typical amplification curve (Ct value
For 15.97), other samples illustrate that this kit has stronger specificity without amplification curve.
Embodiment 6: the detection of clinical sample honeybee Israel acute paralysis virus
10 parts of clinical samples being clinically separated are detected using this kit, it is total conventionally to extract each sample
It is detected after RNA according to the method for embodiment 3.As a result as shown in fig. 6,5 parts of samples are the positive, Ct value 16 ~ 26 it
Between, positive control 16.11, negative control is consistent with expected results without Ct value.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar
<120>a kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box and its detection method
<130> 3
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<170> PatentIn version 3.3
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<213>artificial sequence
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actagtggac gaagcgagtt 20
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tgtactgggc agttacagca 20
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<212> DNA
<213>artificial sequence
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cggaacgccg catgtgttac ca 22
Claims (3)
1. a kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box, it is characterised in that: including forward direction
Primer I APV-F, reverse primer IAPV-R and TaqMan probe IAPV-P, each primer nucleotide sequences are as follows:
Forward primer IAPV-F:5 '-ACTAGTGGACGAAGCGAGTT-3 '；
Reverse primer IAPV-R:5 '-TGTACTGGGCAGTTACAGCA-3 '；
TaqMan probe IAPV-P:5 '-CGGAACGCCGCATGTGTTACCA-3 ',
5 ' the ends and 3 ' ends of probe are respectively adopted fluorophor FAM and BHQ1 and are modified.
2. a kind of honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box according to claim 1,
It is characterized by: the kit includes by following reagent:
(1) RT-PCR reaction solution: the RT-PCR reaction solution constituent of 50 reaction systems are as follows: 5 × One Step RNA PCR
250 μ L of Buffer, concentration are each 25 μ L of forward primer IAPV-F and reverse primer IAPV-R of 10 μm of ol/L, and concentration is 5 μ
The 50 μ L of TaqMan probe IAPV-P of mol/L, concentration are the 25 μ L of dNTP of 10 mmol/L, and concentration is 25 mmol/L's
MgCl2100 μ L, concentration are the 25 μ L of RNase Inhibitor of 40 U/ μ L, amount to 500 μ L；- 20 DEG C of preservations；
(2) Taq DNA polymerase: 25 μ L concentration are 5 U/ μ L Taq DNA polymerase 1, -20 DEG C of preservations；
(3) reverse transcriptase: 25 μ L concentration are 5 U/ μ L reverse transcriptase 1, -20 DEG C of preservations；
(4) positive criteria product: 100 μ L concentration are 1 × 103Copy/μ L positive criteria product 1, using containing target DNA fragment
Positive plasmid as positive criteria product, -20 DEG C of preservations；
(5) negative control: 100 μ L concentration are negative control 1 of 100 ng/ μ L, using the total of healthy honeybee tissue extraction
RNA is negative control sample, -20 DEG C of preservations；
(6) sterile water: 2 mL sterile waters 2.
3. utilizing honeybee Israel acute paralysis virus real-time fluorescent RT-PCR detection reagent box described in as claimed in claim 1 or 22
Detection method, which comprises the following steps:
(1) RT-PCR reaction system configures: it is poly- that RT-PCR reaction solution 10.0 μ L, 5 U/ μ L Taq DNA being added in PCR pipe
Then synthase 0.5 μ L, 5 U/ μ L reverse transcriptase 0.5 μ L, 2.0 μ L of sample to be tested RNA are added 12.0 μ L of sterile water, make
Reaction total volume is 25.0 μ L；
(2) RT-PCR response procedures: 42 DEG C of 15 min of reverse transcription；95 DEG C of 3 min of initial denaturation；Then 95 DEG C of denaturation 5 s, 61
DEG C annealing extend 30 s, totally 40 circulation, in 61 DEG C of progress single-point fluorescence detections；
(3) result judgement: negative control is without Ct value and without amplification curve, while value≤35 positive control Ct, and typical case occurs
Amplification curve, illustrate experiment for effectively experiment, otherwise experiment is invalid；In the case where testing effective situation, sample to be tested is without Ct value
And without amplification curve, indicate in sample without honeybee Israel acute paralysis virus；Value≤35 sample to be tested Ct, and occur typical
Amplification curve, indicate sample in containing honeybee Israel acute paralysis virus；Sample to be tested Ct value > 35, then to the sample weight
Reinspection is surveyed: repeating testing result, then the sample is viral without honeybee Israel acute paralysis without Ct value；Repeating testing result has Ct
It is worth in the then sample containing honeybee Israel acute paralysis virus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112458206A (en) * | 2020-11-25 | 2021-03-09 | 福州海关技术中心 | Primer probe set, application thereof and kit for detecting bee chronic paralysis virus |
| CN112538548A (en) * | 2020-12-04 | 2021-03-23 | 福州海关技术中心 | Primer group and probe for detecting bee Klishmi virus and detection method |
| CN112853003A (en) * | 2021-03-04 | 2021-05-28 | 福州海关技术中心 | Primer group and probe for detecting bee acute paralysis virus, application and detection method thereof |
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| CN105624334A (en) * | 2016-04-18 | 2016-06-01 | 福建出入境检验检疫局检验检疫技术中心 | Bee Israeli acute paralysis virus RT-PCR detection reagent kit and detection method thereof |
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| CN105624334A (en) * | 2016-04-18 | 2016-06-01 | 福建出入境检验检疫局检验检疫技术中心 | Bee Israeli acute paralysis virus RT-PCR detection reagent kit and detection method thereof |
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| PHILIPPE BLANCHARD等: "First detection of Israeli acute paralysis virus (IAPV) in France, a dicistrovirus affecting honeybees (Apis mellifera)", 《J INVERTEBR PATHOL.》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458206A (en) * | 2020-11-25 | 2021-03-09 | 福州海关技术中心 | Primer probe set, application thereof and kit for detecting bee chronic paralysis virus |
| CN112538548A (en) * | 2020-12-04 | 2021-03-23 | 福州海关技术中心 | Primer group and probe for detecting bee Klishmi virus and detection method |
| CN112853003A (en) * | 2021-03-04 | 2021-05-28 | 福州海关技术中心 | Primer group and probe for detecting bee acute paralysis virus, application and detection method thereof |
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| CN109439801B (en) | 2022-03-25 |
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