CN109439801B - Real-time fluorescence RT-PCR detection kit and detection method for israel acute paralysis virus of bees - Google Patents
Real-time fluorescence RT-PCR detection kit and detection method for israel acute paralysis virus of bees Download PDFInfo
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Abstract
The invention relates to a real-time fluorescence RT-PCR detection kit for an Israel acute paralysis virus of bees and a detection method thereof, which are specially used for detecting the Israel acute paralysis virus of the bees. The kit comprises: (1) RT-PCR reaction solution; (2) taq DNA polymerase; (3) reverse transcriptase; (4) a positive standard substance; (5) negative control; (6) sterile water. The invention has the following advantages: (1) good stability and specificity: the kit has high specificity for detecting the israel acute paralysis virus of the bee, has no cross reaction with other bee viruses, and has good repeatability; (2) the sensitivity is high: the sensitivity can reach 10 copies/mu L; (3) the operation is simple, convenient and quick: the entire reaction can be completed in 2 hours. The kit can be used for detecting the israel acute paralysis virus of the bee and the differential diagnosis of other bee virus diseases, and has important significance for early prevention and timely treatment of the disease.
Description
Technical Field
The invention relates to a real-time fluorescence RT-PCR detection kit for an Israel acute paralysis virus of bees and a detection method thereof, belongs to the technical field of animal sanitation, and is suitable for detection of the Israel acute paralysis virus of the bees and differential diagnosis of other bee virus diseases.
Background
Israel Acute Paralysis Virus (IAPV) is a new virus discovered by Ilan Sela et al, university of Hiberella, 2004 as a positive sense single-stranded RNA virus, an unencapsulated icosahedral protein coat, a member of the family Diccistroviridae, members of the order Parapicornaviridae (picorna-like virus). IAPV can infect bee individuals (eggs, larvae, pupae and adult bees) in various developmental stages and all-grade bee colonies (worker bees, male bees and queen bees), and the body color of the affected bees becomes dark, villi falls off, and the affected bees are gradually paralyzed and die along with wing tremor, so that a great deal of bee loss can be caused, and the damage to the bee breeding industry is large. IAPV is widely distributed, and related reports exist in Europe, south America and Asia.
At present, scientists study and analysis show that IAPV (IAPV) is one of main pathogenic factors causing Colony collapse disorder disease (CCD), a large amount of bees disappear, and the loss amount of the bees can reach 80% -100% of the total amount of the whole Colony, so that serious pollination crisis is caused. China is a world-wide apiculture, and the apiculture quantity and the bee product yield are always stable in the first place of the world for many years. The stable development of the bee-keeping industry has important significance for promoting the income increase of farmers, improving the crop yield and maintaining the ecological balance. However, frequent outbreaks of bee diseases have become a major factor affecting the health development of the beekeeping industry in our country, wherein the harm of bee viruses is particularly concerned. Therefore, it is important to establish a method for rapidly and accurately detecting IAPV so as to prevent IAPV infection.
At present, the detection of bee virus diseases mainly adopts immunological detection and nucleic acid molecule detection technologies. The immunity detection is a commonly used method for rapidly detecting the viruses at present, but the viruses of the bees have high gene homology, and the application of the immunology technology can cause poor bee virus detection specificity, so that the accurate diagnosis of the bee virus diseases is difficult. Compared with the immunodetection technology, the PCR detection technology based on nucleic acid molecules has revolutionary success in the aspect of pathogen detection, becomes a gold standard for the rapid detection of nucleic acid, has high sensitivity, strong specificity, simplicity, convenience, rapidness and accuracy, is the most common detection technology at present and is also one of the detection technologies mainly adopted in laboratories. The real-time fluorescent RT-PCR method further enhances the specificity by utilizing the fluorescent probe, directly displays the amplification result without electrophoretic detection, and shortens the detection time.
The disease symptoms of IAPV are similar to those of bee acute paralysis virus (ABPV), the biological and phylogenetic characteristics are very close to those of ABPV and bee keshmir virus (KBV), and the homology of the nucleic acid sequences of the IAPV, the ABPV and the KBV is very high and reaches more than 65%. Sequence alignment and analysis show that the polymerase polyprotein gene has higher specificity compared with ABPV, KBV and IAPV, and the gene is relatively conservative to IAPV, so that the specificity of the method can be improved.
The invention designs primers and probes by utilizing specific conserved sequences of bee Israel acute paralysis virus polymerase polyprotein genes, develops a real-time fluorescent RT-PCR kit and a method for detecting the bee Israel acute paralysis virus by optimizing a reaction system, and can complete the rapid detection of IAPV within 2 h. At present, a real-time fluorescent RT-PCR kit for detecting the israeli acute paralysis virus of the bees and a detection method thereof are not reported, the technology makes up for the technical requirements on bee epidemic disease detection in China at present, and the healthy development of bee colonies and natural ecology in China is protected.
Disclosure of Invention
The invention aims to provide a real-time fluorescence RT-PCR detection kit for the israel acute paralysis virus of bees and a detection method thereof, which make up for the defects of the prior detection technology, have the characteristics of strong specificity, high sensitivity and simple operation, and can quickly and accurately quarantine and identify the israel acute paralysis virus of the bees in bees and bee products.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
a real-time fluorescence RT-PCR detection kit for bee Israel acute paralysis virus is characterized in that: the kit comprises a forward primer IAPV-F, a reverse primer IAPV-R and a TaqMan probe IAPV-P, wherein the nucleotide sequences of the primers are as follows:
forward primer IAPV-F: 5'-ACTAGTGGACGAAGCGAGTT-3' the flow of the air in the air conditioner,
reverse primer IAPV-R: 5'-TGTACTGGGCAGTTACAGCA-3', respectively;
TaqMan probe IAPV-P: 5'-CGGAACGCCGCATGTGTTACCA-3' the flow of the air in the air conditioner,
the 5 'end and the 3' end of the probe are respectively modified by fluorescent groups FAM and BHQ 1.
Wherein, the kit comprises the following reagents:
(1) RT-PCR reaction solution: the RT-PCR reaction solution of 50 reaction systems comprises the following components: 5 Xone Step RNA PCR Buffer 250. mu.L, forward primer IAPV-F and reverse primer IAPV-R each 25. mu.L at a concentration of 10. mu. mol/L, TaqMan probe IAPV-P50. mu.L at a concentration of 5. mu. mol/L, dNTP 25. mu.L at a concentration of 10 mmol/L, MgCl 25 at a concentration of 25 mmol/L 2100 mu L of RNase Inhibitor with the concentration of 40U/mu L, 25 mu L of RNase Inhibitor, and 500 mu L in total; storing at-20 deg.C;
(2) taq DNA polymerase: 25 mu L of Taq DNA polymerase 1 with the concentration of 5U/. mu.L is preserved at-20 ℃;
(3) reverse transcriptase: 25 mul of reverse transcriptase with the concentration of 5U/mul is 1, and the reverse transcriptase is stored at the temperature of minus 20 ℃;
(4) positive standard substance: the concentration of 100. mu.L is 1X 1031 copy/microliter of positive standard substance, adopting positive plasmid containing target DNA fragment as positive standard substance, and storing at-20 deg.C;
(5) negative control: 100 microliter of negative control 1 with the concentration of 100 ng/microliter, adopting total RNA extracted from healthy bee tissue as a negative control sample, and storing at-20 ℃;
(6) sterile water: 2 mL of sterile water.
The invention also aims to provide a detection method using the real-time fluorescence RT-PCR detection kit for the Israeli acute paralysis virus of bees, which comprises the following steps:
(1) and (3) RT-PCR reaction system configuration: adding 10.0 mu L of RT-PCR reaction solution, 0.5 mu L of 5U/mu L Taq DNA polymerase, 0.5 mu L reverse transcriptase and 2.0 mu L RNA of a sample to be detected into a PCR tube, and then adding 12.0 mu L sterilized water to ensure that the total reaction volume is 25.0 mu L;
(2) RT-PCR reaction procedure: reverse transcription is carried out for 15 min at 42 ℃; pre-denaturation at 95 ℃ for 3 min; then, performing denaturation at 95 ℃ for 5 s and annealing and extension at 61 ℃ for 30 s for 40 cycles, and performing single-point fluorescence detection at 61 ℃;
(3) and (4) judging a result: the negative control has no Ct value and no amplification curve, and meanwhile, the positive control Ct value is less than or equal to 35, and a typical amplification curve appears, which indicates that the experiment is effective, otherwise, the experiment is ineffective; under the condition that the experiment is effective, the sample to be detected has no Ct value and no amplification curve, and the sample does not contain the Israel acute paralysis virus; ct value of the sample to be detected is less than or equal to 35, and a typical amplification curve appears, which indicates that the sample contains Israel acute paralysis viruses of bees; if the Ct value of the sample to be detected is more than 35, repeatedly detecting the sample: if the repeated detection result has no Ct value, the sample does not contain the Israel acute paralysis virus of the bee; if the repeated detection result has a Ct value, the sample contains the israeli acute paralysis virus of the bee.
The invention has the following advantages: (1) good stability and specificity: the invention selects the conserved segment of the bee Israel acute paralysis virus polymerase polyprotein gene as a target, not only designs a pair of specific primers, but also designs a specific fluorescent probe, can carry out double control on the target in the fluorescent RT-PCR reaction process, has high specificity for detecting the bee Israel acute paralysis virus, has no cross reaction with other bee viruses, and has good repeatability; (2) the sensitivity is high: the invention adopts a TaqMan-BHQ1 probe, and further improves the sensitivity of the probe by optimizing a reaction system and reaction conditions, and the sensitivity can reach 10 copies/mu L; (3) the operation is simple, convenient and quick: data are collected by real-time fluorescence, electrophoresis is not needed for detecting the amplification condition of nucleic acid, and the whole reaction can be completed within 2 hours.
Drawings
FIG. 1 shows the results of RT-PCR amplification of positive standard and negative control in example 1. Wherein M: 100bp Ladder DNA Marker I; 1: a positive standard substance with the size of about 127 bp; 2: and (5) negative control.
FIG. 2 shows the annealing temperature optimization results of the detection method of the real-time fluorescence RT-PCR detection kit for Israel acute paralysis virus in example 2. When the annealing temperature is 61.4 ℃, the fluorescence intensity is strongest, so the optimal annealing temperature in the detection method of the kit is 61 ℃.
FIG. 3 is the kinetic curve of fluorescent RT-PCR of the positive standard for the Israel acute paralysis Virus of the bee of example 4. The abscissa in the figure represents the cycle number of fluorescent RT-PCR amplification and the ordinate represents the fluorescent signal intensity; the concentrations of the positive standards of the right-to-left amplification curves were 1X 10, respectively1To 1X 108Copies/. mu.L.
FIG. 4 is a standard curve of real-time fluorescent RT-PCR of the positive standard for the bee Israel acute paralysis virus of example 4. In the figure, the abscissa represents the copy number of the standard substance, and the ordinate represents the number of RT-PCR amplification cycles; the standard curve equation is Y = -3.479x + 39.841; r represents a correlation coefficient, and the coefficient R is determined20.973, the amplification efficiency was 98.359%.
FIG. 5 shows the specific assay result of the real-time fluorescent RT-PCR detection kit for the bee Israel acute paralysis virus in example 5. Wherein 1: israeli acute paralysis virus of bee; 2: black bee queen cell virus; 3: bee sacbrood virus; 4: acute paralysis virus; 5: a residual wing virus; 6: and (5) negative control.
FIG. 6 shows the results of clinical sample testing in example 6. Positive controls, negative controls and 5 positive samples were included.
Detailed Description
In order to further illustrate the invention, but not to limit it, reference is made to the following examples. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials are commercially available without specific mention.
Example 1:
a real-time fluorescence RT-PCR detection kit for an Israeli acute paralysis virus of bees comprises a forward primer IAPV-F, a reverse primer IAPV-R and a TaqMan probe IAPV-P, wherein the sequence of the forward primer IAPV-F is 5'-ACTAGTGGACGAAGCGAGTT-3', and the sequence of the reverse primer IAPV-R is 5'-TGTACTGGGCAGTTACAGCA-3'; the IAPV-P sequence of the TaqMan probe is 5 '-FAM-CGGAACGCCGCATGTGTTACCA-BHQ 1-3'. The kit comprises the following reagents (50 reaction systems):
(1) RT-PCR reaction solution: the RT-PCR reaction solution of 50 reaction systems comprises the following components: 250 mu L of 5 xOne Step RNA PCR Buffer, 25 mu L of each of a forward primer IAPV-F and a reverse primer IAPV-R with the concentration of 10 mu mol/L, 50 mu L of a TaqMan probe IAPV-P with the concentration of 5 mu mol/L, 25 mu L of dNTP with the concentration of 10 mmol/L, 2100 mu L of MgCl with the concentration of 25 mmol/L and 25 mu L of RNase Inhibitor with the concentration of 40U/mu L, and the total volume is 500 mu L; storing at-20 deg.C;
(2) taq DNA polymerase: 25 mu L of Taq DNA polymerase 1 with the concentration of 5U/. mu.L is preserved at-20 ℃;
(3) reverse transcriptase: 25 mul of reverse transcriptase with the concentration of 5U/mul is 1, and the reverse transcriptase is stored at the temperature of minus 20 ℃;
(4) positive standard substance: the concentration of 100. mu.L is 1X 1031 copy/microliter of positive standard substance, adopting positive plasmid containing target DNA fragment as positive standard substance, and storing at-20 deg.C;
(5) negative control: 100 microliter of negative control 1 with the concentration of 100 ng/microliter, adopting total RNA extracted from healthy bee tissue as a negative control sample, and storing at-20 ℃;
(6) sterile water: 2 mL of sterile water.
Example 2:
annealing temperature optimization of detection method of real-time fluorescence RT-PCR detection kit for Israeli bee acute paralysis virus
(1) RT-PCR reaction system: respectively adding 10.0 mu L of RT-PCR reaction liquid, 0.5 mu L of 5U/mu L Taq DNA polymerase, 0.5 mu L of 5U/mu L reverse transcriptase and 2.0 mu L of standard sample diluted by 10 times in an increasing way into each PCR tube, and then adding 12.0 mu L of sterilized water to ensure that the total reaction volume is 25.0 mu L;
(2) RT-PCR optimization reaction program: reverse transcription is carried out for 15 min at 42 ℃; pre-denaturation at 95 ℃ for 3 min; then denaturation at 95 ℃ for 5 s, annealing at 55-65 ℃ (gradient temperature) for 30 s, setting 3 repetitions per gradient temperature, and repeating for 40 cycles;
(3) selecting annealing temperature: the result of the annealing temperature optimization is shown in FIG. 2, and the annealing temperature is 61.4 ℃ because the fluorescence intensity of the amplification product is the highest, 61 ℃ is selected as the annealing temperature of the reaction program of the kit.
Example 3:
the detection method using the real-time fluorescence RT-PCR detection kit for the Israeli bee acute paralysis virus comprises the following steps:
(1) and (3) RT-PCR reaction system configuration: adding 10.0 mu L of RT-PCR reaction solution, 0.5 mu L of 5U/mu L Taq DNA polymerase, 0.5 mu L reverse transcriptase and 2.0 mu L RNA of a sample to be detected into a PCR tube, and then adding 12.0 mu L sterilized water to ensure that the total reaction volume is 25.0 mu L;
(2) RT-PCR reaction procedure: reverse transcription is carried out for 15 min at 42 ℃; pre-denaturation at 95 ℃ for 3 min; then, performing denaturation at 95 ℃ for 5 s and annealing and extension at 61 ℃ for 30 s for 40 cycles, and performing single-point fluorescence detection at 61 ℃;
(3) and (4) judging a result: the negative control has no Ct value and no amplification curve, and meanwhile, the positive control Ct value is less than or equal to 35, and a typical amplification curve appears, which indicates that the experiment is effective, otherwise, the experiment is ineffective; under the condition that the experiment is effective, the sample to be detected has no Ct value and no amplification curve, and the sample does not contain the Israel acute paralysis virus; ct value of the sample to be detected is less than or equal to 35, and a typical amplification curve appears, which indicates that the sample contains Israel acute paralysis viruses of bees; if the Ct value of the sample to be detected is more than 35, repeatedly detecting the sample: if the repeated detection result has no Ct value, the sample does not contain the Israel acute paralysis virus of the bee; if the repeated detection result has a Ct value, the sample contains the israeli acute paralysis virus of the bee.
Example 4:
establishing a detection standard curve of the real-time fluorescence RT-PCR detection kit for the Israel acute paralysis viruses of the bees:
(1) diluting the standard product DNA: the positive cloning plasmid containing the target fragment was diluted 10-fold incrementally with sterile water to 1X 108Copies/. mu.L-1X 101Copies/. mu.L of a series of plasmid DNA standards;
(2) establishing a standard curve equation: the diluted plasmid DNA standard is used as a template, each standard sample is processed and repeated for 3 times, the total RNA of the healthy bee tissue is used as a control, and sterile water is used as a blank control.Configuring a 25 mu L real-time fluorescent RT-PCR reaction system and carrying out real-time fluorescent RT-PCR reaction according to the mode of the embodiment 3; an amplification curve as shown in FIG. 3 was obtained, and then a standard curve as shown in FIG. 4 was plotted to construct a standard curve equation. The standard curve equation constructed in this example is Y = -3.479x + 39.841; r represents a correlation coefficient, and the coefficient R is determined20.973, the amplification efficiency was 98.359%.
Example 5: specificity determination of real-time fluorescence RT-PCR detection kit for Israeli bee acute paralysis virus
5 common bee viruses such as the israel acute paralysis virus, the queen bee virus, the bee sacbrood virus, the acute paralysis virus, the residual wing virus and the like are respectively used as samples, RNA is extracted according to a conventional method, and then RT-PCR reaction and result judgment are carried out by the method of the embodiment 3, as can be seen from figure 5, only the israel acute paralysis virus of the bee generates a typical amplification curve (Ct value is 15.97), and other samples have no amplification curve, which indicates that the kit has stronger specificity.
Example 6: detection of clinical sample bee Israeli acute paralysis virus
The kit is used for detecting 10 clinical samples separated clinically, and the total RNA of each sample is extracted according to a conventional method and then detected according to the method of the embodiment 3. The results are shown in FIG. 6, 5 samples are positive, Ct values are all between 16 and 26, the positive control is 16.11, and the negative control has no Ct value and is consistent with the expected results.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> inspection and quarantine technology center of Fujian entry-exit inspection and quarantine bureau
<120> real-time fluorescence RT-PCR detection kit for israel acute paralysis virus of honeybees and detection method thereof
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<170> PatentIn version 3.3
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<212> DNA
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cggaacgccg catgtgttac ca 22
Claims (1)
1. A real-time fluorescence RT-PCR detection kit for bee Israel acute paralysis virus is characterized in that: the kit comprises a forward primer IAPV-F, a reverse primer IAPV-R and a TaqMan probe IAPV-P, wherein the nucleotide sequences of the primers are as follows:
forward primer IAPV-F: 5'-ACTAGTGGACGAAGCGAGTT-3', respectively;
reverse primer IAPV-R: 5'-TGTACTGGGCAGTTACAGCA-3', respectively;
TaqMan probe IAPV-P: 5'-CGGAACGCCGCATGTGTTACCA-3' the flow of the air in the air conditioner,
the 5 'end and the 3' end of the probe are respectively modified by fluorescent groups FAM and BHQ 1;
the kit comprises the following reagents:
(1) RT-PCR reaction solution: the RT-PCR reaction solution of 50 reaction systems comprises the following components: 5 Xone Step RNA PCR Buffer 250. mu.L, forward primer IAPV-F and reverse primer IAPV-R each 25. mu.L at a concentration of 10. mu. mol/L, TaqMan probe IAPV-P50. mu.L at a concentration of 5. mu. mol/L, dNTP 25. mu.L at a concentration of 10 mmol/L, MgCl 25 at a concentration of 25 mmol/L2100 mu L of RNase Inhibitor with the concentration of 40U/mu L, 25 mu L of RNase Inhibitor, and 500 mu L in total; storing at-20 deg.C;
(2) taq DNA polymerase: 25 mu L of Taq DNA polymerase 1 with the concentration of 5U/. mu.L is preserved at-20 ℃;
(3) reverse transcriptase: 25 mul of reverse transcriptase with the concentration of 5U/mul is 1, and the reverse transcriptase is stored at the temperature of minus 20 ℃;
(4) positive standard substance: the concentration of 100. mu.L is 1X 1031 copy/microliter of positive standard substance, adopting positive plasmid containing target DNA fragment as positive standard substance, and storing at-20 deg.C;
(5) negative control: 100 microliter of negative control 1 with the concentration of 100 ng/microliter, adopting total RNA extracted from healthy bee tissue as a negative control sample, and storing at-20 ℃;
(6) sterile water: 2 pieces of 2 mL sterile water;
the use method of the kit comprises the following steps:
(1) and (3) RT-PCR reaction system configuration: adding 10.0 mu L of RT-PCR reaction liquid, 0.5 mu L of 5U/mu L Taq DNA polymerase, 0.5 mu L reverse transcriptase and 2.0 mu L RNA of a sample to be detected into a PCR tube, and then adding 12.0 mu L sterile water to ensure that the total reaction volume is 25.0 mu L;
(2) RT-PCR reaction procedure: reverse transcription is carried out for 15 min at 42 ℃; pre-denaturation at 95 ℃ for 3 min; then, performing denaturation at 95 ℃ for 5 s and annealing and extension at 61 ℃ for 30 s for 40 cycles, and performing single-point fluorescence detection at 61 ℃;
(3) and (4) judging a result: the negative control has no Ct value and no amplification curve, and meanwhile, the positive control Ct value is less than or equal to 35, and a typical amplification curve appears, which indicates that the experiment is effective, otherwise, the experiment is ineffective; under the condition that the experiment is effective, the sample to be detected has no Ct value and no amplification curve, and the sample does not contain the Israel acute paralysis virus; ct value of the sample to be detected is less than or equal to 35, and a typical amplification curve appears, which indicates that the sample contains Israel acute paralysis viruses of bees; if the Ct value of the sample to be detected is more than 35, repeatedly detecting the sample: if the repeated detection result has no Ct value, the sample does not contain the Israel acute paralysis virus of the bee; if the repeated detection result has a Ct value, the sample contains the israeli acute paralysis virus of the bee.
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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 |
Non-Patent Citations (3)
| Title |
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| First detection of Israeli acute paralysis virus (IAPV) in France, a dicistrovirus affecting honeybees (Apis mellifera);Philippe Blanchard等;《J Invertebr Pathol.》;20080730;第99卷(第3期);348-350 * |
| 以色列急性麻痹病毒（IAPV）RT-PCR检测方法的建立及应用;张体银等;《中国兽医学报》;20180630;第38卷(第6期);1105-1108 * |
| 张体银等.以色列急性麻痹病毒（IAPV）RT-PCR检测方法的建立及应用.《中国兽医学报》.2018,第38卷(第6期),1105-1108. * |
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