CN111979340B - Specific primer and probe for identifying Bactrocera dorsalis and application of specific primer and probe - Google Patents
Specific primer and probe for identifying Bactrocera dorsalis and application of specific primer and probe Download PDFInfo
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Abstract
The invention relates to a specific primer and a probe for identifying Bactrocera dorsalis and application thereof. The sequence of the specific primer comprises a sequence shown in SEQ ID NO.1 and a sequence shown in SEQ ID NO. 2. The sequence of the probe comprises the sequence shown in SEQ ID NO. 3. The molecular biological quarantine identification method for rapidly detecting the Bactrocera dorsalis built by the primer and/or the probe has the advantages of high detection rate, short quarantine time, strong specificity, high sensitivity and the like.
Description
Technical Field
The invention belongs to the technical field of insect quarantine identification, and particularly relates to a specific primer and a probe for identifying Bactrocera dorsalis and application thereof.
Background
The European cherry fruit fly Rhagoletics ras (L.) belongs to the genus Diptera of the order Tephritidae, the family of the genus Tephritidae, around the genus Bactria Rhagoletis. The around-fruit fly genus (non-Chinese species) is a quarantine pest prohibited from entering into the environment in China specified in the "people's republic of China's entry plant quarantine pest directory".
Bactrocera dorsalis is mainly distributed in Europe, asia and other areas, is a highly destructive insect, and seriously damages cherry industry in Europe, asia and other areas. If chemical control is not carried out in Europe, the infection rate can reach up to 100 percent, and the yield loss can reach up to 60 to 100 percent. The fruit fly host plant is wide and comprises various kinds of plum plants (Rosaceae, sour cherry, sweet cherry, black cherry, mahali cherry), lonicera plants (Caprifoliaceae, lonicera japonica) and berberidaceae plants (Berberis). In addition, the control work of the Bactrocera dorsalis is very difficult, and no effective method for controlling the Bactrocera dorsalis is found up to now.
The Eurotium cherry fly is harmful organism required to be paid attention by documents such as the Inlet Utzbexakistan cherry, uyerba orange and Chili avocado plant inspection and quarantine requirement (No. 55 in 2017), the Inlet New Zealand persimmon and Turkish cherry plant inspection and quarantine requirement (No. 51 in 2016), the Inlet Tajikistan cherry plant inspection and quarantine requirement (No. 79 in 2013) and the like. In 2017-2018, capturing the fruit fly larvae in cherry fruits carried by passengers for many times at the mouth of the Guangzhou airport and the bead sea; the Urufimbriae Portland also captures the Bactrocera larvae in the Gilgias imported cherry. The general office of quality inspection in the original country issued "warning notification about the illegal entry of the European cherry fly with the passenger's carried object" in 2017 (quality inspection police [2017] No. 038). In addition, most of northern areas in China are in temperate areas, and the latitude of the distribution of the cherry fruit flies is consistent with that of the cherry fruit flies in Europe, so that the cherry fruit flies are presumed to have a wide range in the suitable living areas in China. In addition, the host plants of the European cherry fruit flies such as cherry, honeysuckle and the like are widely planted in China, and in 2016, china exceeds Turkey, so that the plant becomes the first cherry production country in the world. The lonicera plant is an important water and soil conservation, landscaping and medicinal cultivation variety in China, and has huge planting area and higher economic value. Therefore, once the fruit fly is transferred into China, the fruit fly can seriously strike cherry industry, landscaping industry and other industries in China, and the export trade of fruits in China can be seriously influenced, so that huge economic loss is brought. The method has important significance for protecting ecological safety of agriculture and forestry production in China, effectively preventing invasion colonization and transmission of the pests and enhancing research on a rapid quarantine identification method of the pests.
At present, aiming at quarantine identification of the European cherry fruit fly, the inspection and quarantine departments of the entry and exit ports still mainly rely on morphological characteristics of adult fruit flies for identification, and the identification of other insect states such as larvae is always a difficult problem. In most cases, the larvae of the fruit flies are the most main larvae interception states, and the morphological characteristics of most larvae of the fruit flies are generally similar, and the types of the larvae are difficult to distinguish through the morphological characteristics, so that port inspection and quarantine departments generally adopt a molecular biological method to identify the larvae. The larval identification steps by using the universal primer sequencing method are complex and take a long time: (1) extracting DNA from the captured fly larvae; (2) PCR detection of the extracted DNA with common primers of insect COI gene, etc.; (3) Detecting whether a target band is obtained by gel electrophoresis of the PCR product; (4) After detecting and obtaining a target strip, sending the PCR product to a sequencing company for sequencing, and waiting for a sequencing result; (5) And (3) performing Blast sequence comparison on the sequencing result on the NCBI website, and judging whether the sequencing result is Bactrocera dorsalis after analyzing the sequence comparison result. This process often requires 3-7 days to complete, and cannot meet the working requirements of rapid, accurate and sensitive identification of the daily port.
At present, a rapid quarantine identification method for the Bactrocera dorsalis has no method, and has no corresponding national standard and industry standard. Therefore, the rapid and accurate detection of the Bactrocera dorsalis is an urgent problem to be solved.
Disclosure of Invention
In view of the problems existing in the prior art, the invention provides a specific primer and a probe for identifying Bactrocera dorsalis and application thereof. The molecular biological quarantine identification method for rapidly detecting the Bactrocera dorsalis built by the primer and/or the probe has the advantages of high detection rate, short quarantine time, strong specificity, high sensitivity and the like.
The technical scheme for solving the technical problems is as follows:
a specific primer for identifying Bactrocera dorsalis, the sequence of which comprises the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO. 2. The primer comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer comprises the sequence shown in SEQ ID NO.1, and the sequence of the downstream primer comprises the sequence shown in SEQ ID NO. 2.
The beneficial effects of the invention include: the molecular biological quarantine identification method for rapidly detecting the Bactrocera dorsalis established by the primers has the advantages of high detection rate, short quarantine time, strong specificity, high sensitivity and the like.
Preferably, the sequence of the upstream primer is the sequence shown as SEQ ID NO.1, and the sequence of the downstream primer is the sequence shown as SEQ ID NO. 2.
The inventors, at the time of primer design, selected a region containing a specific site of Bactrocera dorsalis to design a primer, and found a primer pair capable of specifically amplifying Bactrocera dorsalis by evaluating primer dimer, hairpin structure, mismatch priming efficiency, annealing temperature of upstream and downstream primers, etc., in a region containing the specific site. Meanwhile, in order to improve the specificity, the specificity locus is ensured to be positioned at the 3' end of the primer as much as possible, and the annealing temperature is improved. Therefore, all factors are comprehensively considered, the species-specific primer meeting all conditions is designed, and the primer is verified by multiple tests, so that the unexpected discovery has the advantages of high detection rate, short quarantine time, strong specificity, high sensitivity and the like.
The invention provides a probe for identifying Bactrocera dorsalis, and the sequence of the probe comprises a sequence shown as SEQ ID NO. 3. Preferably, the sequence of the probe is the sequence shown in SEQ ID NO. 3.
The beneficial effects of the invention include: the probe can be used for molecular biological quarantine identification of cherry fruit flies and has the advantages of high detection rate, short quarantine time, strong specificity, high sensitivity and the like.
The inventor comprehensively considers all factors when designing a probe sequence, selects a region containing a specific site of the Bactrocera dorsalis to design the probe, and finds the probe capable of specifically binding the Bactrocera dorsalis by evaluating the dimer, hairpin structure, mismatch initiation efficiency and the like of the probe within the region containing the specific site, and simultaneously combining the characteristics of specific primers, so that the annealing temperature of the probe is higher than the annealing temperature of the primers. By designing the species-specific probes meeting all conditions and carrying out multiple times of verification of experiments, the unexpected discovery that the probes are adopted for detection has the advantages of high detection rate, short quarantine time, strong specificity, high sensitivity and the like.
Further, the probe is also labeled with a fluorescent labeling group. For example, FAM (6-carboxyfluorescein) and BHQ1 are labeled at the 5 'and 3' ends of the probe, respectively, and the use of the above groups is advantageous for improving sensitivity.
The invention provides application of the primer and/or the probe in identification of the Bactrocera dorsalis.
The invention provides a method for identifying Bactrocera dorsalis, which comprises the following steps: and carrying out PCR amplification on the DNA of the sample to be identified by using the primer.
The identification method has the advantages of high detection rate, short quarantine time, strong specificity, high sensitivity and the like.
Specifically, the reaction system for PCR amplification may include: 10 XPCR buffer 2.5. Mu.L, 50mmol/L magnesium chloride 2.0. Mu.L, 10mmol/L dNTP 2.0. Mu.L, 10. Mu.mol/L upstream primer 1.0. Mu.L, 10. Mu.mol/L downstream primer 1.0. Mu.L, 5U/. Mu.L Taq DNA polymerase 0.5. Mu.L, 10-100 ng/. Mu.L template DNA 1.0. Mu.L, ddH 2 O was made up to 25. Mu.L of the entire reaction system.
Further, the reaction conditions for PCR amplification may include: pre-denaturation: 3min at 95 ℃; denaturation: annealing at 95 ℃ for 15 s: extension at 65℃for 1 min: 30s at 72 ℃ for 30 cycles; extension: and at 72℃for 10min.
The reaction condition of PCR amplification is favorable for the amplification of the Bactrocera dorsalis specific primer, and the annealing temperature can greatly reduce the non-specific combination between the primer and the template and improve the specificity of the PCR reaction.
The invention provides a method for identifying Bactrocera dorsalis, which comprises the following steps: and carrying out fluorescent quantitative PCR amplification on the DNA of the sample to be identified by using the primer and the probe.
Specifically, the fluorescent quantitative PCR amplification reaction system may include: 2 XTaqMan real-time PCR master mix 12.5.5. Mu.L, 10. Mu. Mol/L upstream primer 0.5. Mu.L, 10. Mu. Mol/L downstream primer 0.5. Mu.L, 10. Mu. Mol/L probe 0.5. Mu.L, 10-100 ng/. Mu.L template DNA 1. Mu.L, ddH 2 O was added to 25. Mu.L of the PCR amplification reaction system.
Further, the reaction conditions for fluorescent quantitative PCR amplification include: 95 ℃ for 30s; collecting fluorescent signals after each cycle is finished in 95 ℃ for 5s and 65 ℃ for 35s and 40 cycles; 60 ℃ for 1min.
The reaction conditions of the fluorescent quantitative PCR amplification are favorable for the amplification of the Bactrocera dorsalis specific primers.
The invention provides a kit for identifying Bactrocera dorsalis, which comprises the primer and/or the probe.
Further, the kit also comprises one or a combination of more of PCR amplification buffer solution, magnesium chloride, dNTPs, enzyme for common PCR amplification, sterile water for fluorescent quantitative PCR amplification, template DNA to be detected, negative control and positive control.
For example: the PCR detection kit for identifying Bactrocera dorsalis may comprise: 10 XPCR buffer, 50mmol/L magnesium chloride, 10mmol/L dNTP, 10. Mu. Mol/L upstream primer, 10. Mu. Mol/L downstream primer, 5U/. Mu.L Taq DNA polymerase, 10-100 ng/. Mu.L template DNA, ddH 2 O, etc.
The fluorescent quantitative PCR assay kit for identifying bactrocera dorsalis may comprise: 2 XTaqMan real-time PCR master mix, 10. Mu. Mol/L upstream primer, 10. Mu. Mol/L downstream primer, 10. Mu. Mol/L probe, 10-100 ng/. Mu.L template DNA, ddH 2 O, etc.
The invention provides application of the kit in identifying Bactrocera dorsalis.
The method for identifying the Bactrocera dorsalis using the PCR detection kit can comprise: and carrying out PCR amplification on the DNA of the sample to be identified by adopting the kit. The reaction system for PCR amplification may include: 10 XPCR buffer 2.5. Mu.L, 50mmol/L magnesium chloride 2.0. Mu.L, 10mmol/L dNTP 2.0. Mu.L, 10. Mu.mol/L upstream primer 1.0. Mu.L, 10. Mu.mol/L downstream primer 1.0. Mu.L, 5U/. Mu.L Taq DNA polymerase 0.5. Mu.L, 10-100 ng/. Mu.L template DNA 1.0. Mu.L, ddH 2 O was made up to 25. Mu.L of the entire reaction system. The reaction conditions for PCR amplification may include: pre-denaturation: 3min at 95 ℃; denaturation: annealing at 95 ℃ for 15 s: extension at 65℃for 1 min: 30s at 72 ℃ for 30 cycles; extension: and at 72℃for 10min.
The method for identifying the Bactrocera dorsalis by using the fluorescent quantitative PCR detection kit can comprise the following steps: and carrying out fluorescent quantitative PCR amplification on the DNA of the sample to be identified by adopting the kit. The fluorescent quantitative PCR amplification reaction system can comprise: 2 XTaqMan real-time PCR master mix 12.5.5. Mu.L, 10. Mu. Mol/L upstream primer 0.5. Mu.L, 10. Mu. Mol/L downstream primer 0.5. Mu.L, 10. Mu. Mol/L probe 0.5. Mu.L, 10-100 ng/. Mu.L template DNA 1-2. Mu.L, ddH 2 O was added to 25. Mu.L of the PCR amplification reaction system. The reaction conditions for fluorescent quantitative PCR amplification include: 95 ℃ for 30s; collecting fluorescent signals after each cycle is finished in 95 ℃ for 5s and 65 ℃ for 35s and 40 cycles; 60 ℃ for 1min.
Drawings
FIG. 1 shows the results of specific test for Bactrocera dorsalis in example 3. Wherein, M is DL1200 DNA Marker;1: bactrocera dorsalis; 2: bactrocera dorsalis; 3: bactrocera dorsalis; 4: fruit fly; 5: three-point bactrocera dorsalis; 6: melon fly; 7: citrus fruit fly; 8: fruit fly in Mediterranean; 9: guava fruit fly; 10: sea buckthorn winds around the fruit fly; 11: pumpkin fruit fly; 12: fruit fly of south America; 13: fruit fly with stripe; 14: ddH 2 O。
FIG. 2 shows the result of primer sensitivity verification test for cherry fruit fly in example 3. Wherein, M is DL600 DNA Marker;1: a DNA stock solution; 2:10 -1 ；3：10 -2 ；4：10 -3 ；5：10 -4 ；6：10 -5 ；7：ddH 2 O。
FIG. 3 is the result of primer specificity verification in example 4.
FIG. 4 is the result of primer sensitivity verification in example 4.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
The invention provides a specific primer and a detection method for rapidly identifying Bactrocera dorsalis.
The invention designs a group of specific primers for rapidly identifying the Bactrocera dorsalis, and the sequences of the primers are as follows:
upstream primer OYF:5'-GTAATTGTTACAGCCCATGCC-3' (SEQ ID NO. 1);
downstream primer OYR:5'-GTAAACAGTTCAACCTGTC-3' (SEQ ID NO. 2).
The above primers were synthesized by Shanghai Siborui Biotechnology Co.
The invention also provides a PCR detection method for rapidly quarantining and identifying the Bactrocera dorsalis larvae, which sequentially comprises the following steps:
(1) Extraction of sample DNA to be detected
Placing a fruit fly sample to be detected into a 1.5ml centrifuge tube, mashing or adding liquid nitrogen for grinding, and extracting DNA by using a commercial kit.
(2) Ordinary PCR amplification
A. 25. Mu.L of PCR reaction system was used:
| composition of the composition | Sample addition amount/. |
| 10 XPCR buffer | 2.5 |
| 50mmol/L magnesium chloride | 2.0 |
| 10mmol/LdNTP | 2.0 |
| 10 mu mol/L primer OYF | 1.0 |
| 10 mu mol/L primer OYR | 1.0 |
| 5U/. Mu.LTaqDNA polymerase | 0.5 |
| 10-100 ng/. Mu.L template DNA | 1.0 |
| ddH 2 O | Supplement to 25 |
Note that: the amount of each reagent in the reaction system can be appropriately adjusted according to the specific conditions or different total reaction volumes.
The components in the reaction system are purchased from the division of biological engineering (Shanghai).
B. The PCR reaction tube is put into a common PCR instrument, and PCR amplification is completed according to the following reaction conditions:
the PCR reaction conditions were: pre-denaturation: 3min at 95 ℃; (denaturation: 95 ℃ C. For 15s; annealing: 65 ℃ C. For 1min; extension: 72 ℃ C. For 30 s) 30 cycles; extension: and at 72℃for 10min.
C. Judging according to the amplification result, if the detected sample has 222bp expected amplified fragment, judging as positive, if the detected sample has 222bp expected amplified fragment, judging as negative.
The invention also designs a group of fluorescent quantitative PCR primers and probes (OYP) for rapidly identifying the Bactrocera dorsalis, and the sequences of the primers and the probes are as follows:
OYF：5’-GTAATTGTTACAGCCCATGCC-3’(SEQ ID NO.1)；
OYR：5’-GTAAACAGTTCAACCTGTC-3’(SEQ ID NO.2)。
OYP: the sequence of the 5'-FAM-AGGAGCACCAGATATAGCTTTTCCTCG-BHQ1-3' probe OYP is shown in SEQ ID NO. 3.
The probe was synthesized by Shanghai Sideri Biotechnology Co.
The invention also provides a fluorescence quantitative PCR detection method for rapidly quarantining and identifying the Bactrocera dorsalis, which sequentially comprises the following steps:
(1) Extraction of sample DNA to be detected
The Bactrocera dorsalis sample is placed in a 1.5ml centrifuge tube, smashed or added with liquid nitrogen for grinding, and DNA extraction is carried out by adopting a commercial kit.
(2) Fluorescent quantitative PCR amplification
A. A 25. Mu.L fluorescent quantitative PCR reaction system was used:
the components in the reaction system are purchased from the division of biological engineering (Shanghai).
The PCR reaction tube is put into a fluorescent quantitative PCR instrument, and PCR amplification is completed according to the following reaction conditions:
the fluorescent quantitative reaction conditions are as follows: 95 ℃ for 30s; collecting fluorescent signals after each cycle is finished in 95 ℃ for 5s and 65 ℃ for 35s and 40 cycles; 60 ℃ for 1min.
Judging according to the result of fluorescent quantitative PCR amplification, and judging positive if the Ct value of the detection sample is less than or equal to 35; if the template concentration is required to be adjusted between 35 and 40 (namely the CT value is more than 35 and less than or equal to 40), repeating the test, and judging that the Ct value of the repeated result is still between 35 and 40, and if the Ct value is still positive, judging that the Ct value is negative; the detection sample is not amplified or Ct value is more than 40, and the detection sample is judged as negative.
The invention has the beneficial effects that: the invention establishes a molecular biological quarantine identification method for rapidly detecting the Bactrocera dorsalis, establishes standards and bases for quarantining the Bactrocera dorsalis, is beneficial to strengthening port quarantine strength of the Bactrocera dorsalis, improves quarantine rate, shortens quarantine time, prevents the Bactrocera dorsalis from being transmitted into China, and avoids diffusion and propagation of the Bactrocera dorsalis in China.
Compared with the traditional morphological identification method and the general primer sequencing method, the method established by the invention has stronger specificity and higher sensitivity, and the DNA sensitivity of the primer identification is about 0.001 ng/. Mu.L.
The following description is made by way of specific examples.
Example 1
The present example provides a set of specific primers for rapid identification of Bactrocera dorsalis, the sequences of the primers are as follows:
OYF：5’-GTAATTGTTACAGCCCATGCC-3’(SEQ ID NO.1)；
OYR：5’-GTAAACAGTTCAACCTGTC-3’(SEQ ID NO.2)。
the invention also provides a PCR detection method for rapidly quarantining and identifying the Bactrocera dorsalis larvae, which sequentially comprises the following steps:
1. extraction of sample DNA to be detected
Placing a fruit fly sample into a 1.5ml centrifuge tube, wherein the fruit fly sample can be adults, larvae or pupae, mashing or adding liquid nitrogen for grinding, and extracting DNA by using a commercial kit.
2. Ordinary PCR amplification
A. 25. Mu.L of PCR reaction system was used:
B. the PCR reaction tube is put into a common PCR instrument, and PCR amplification is completed according to the following reaction conditions:
the PCR reaction conditions were: pre-denaturation: 3min at 95 ℃; (denaturation: 95 ℃ C. For 15s; annealing: 65 ℃ C. For 1min; extension: 72 ℃ C. For 30 s) 30 cycles; extension: and at 72℃for 10min.
If the detected sample has 222bp expected amplified fragment, the detection sample is judged to be positive, and if the detected sample has no 222bp expected amplified fragment, the detection sample is judged to be negative.
Example 2
The invention also designs a group of fluorescent quantitative PCR primers and probes for rapidly identifying the Bactrocera dorsalis, and the sequences of the primers and the probes are as follows:
OYF：5’-GTAATTGTTACAGCCCATGCC-3’(SEQ ID NO.1)；
OYR：5’-GTAAACAGTTCAACCTGTC-3’(SEQ ID NO.2)；
OYP:5'-FAM-AGGAGCACCAGATATAGCTTTTCCTCG-BHQ1-3' (probe sequence shown in SEQ ID NO. 3).
The invention also provides a fluorescent quantitative PCR detection method for rapidly quarantining and identifying the Bactrocera dorsalis larvae, which sequentially comprises the following steps:
1. DNA extraction of sample to be detected
Placing a fruit fly sample into a 1.5ml centrifuge tube, wherein the fruit fly sample can be adults, larvae or pupae, mashing or adding liquid nitrogen for grinding, and extracting DNA by using a commercial kit.
2. Fluorescent quantitative PCR amplification
A. A 25. Mu.L fluorescent quantitative PCR reaction system was used:
| composition of the composition | Sample addition amount/. |
| 2×TaqMan real-time PCR master mix | 12.5 |
| 10 mu mol/L primer OYF | 0.5 |
| 10 mu mol/L primer OYR | 0.5 |
| 10 mu mol/L Probe OYP | 0.5 |
| 10-100ng/ |
1 |
| ddH 2 O | Is added to 25 mu L |
3. The PCR reaction tube is put into a fluorescent quantitative PCR instrument, and PCR amplification is completed according to the following reaction conditions:
the fluorescent quantitative reaction conditions are as follows: 95 ℃ for 30s; collecting fluorescent signals after each cycle is finished in 95 ℃ for 5s and 65 ℃ for 35s and 40 cycles; 60 ℃ for 1min. (A plurality of fluorescence quantitative PCR instruments such as ABI 7500fast, ABI Quantum studio 6 Flex and the like can be used for fluorescence quantitative PCR amplification, and when different instruments and reagents are used, the reaction parameters can be properly adjusted according to requirements).
The test result shows that the Ct value of the test sample is less than or equal to 35 and is judged to be positive; if the CT value is between 35 and 40 (namely the CT value is more than 35 and less than or equal to 40), the template concentration is required to be adjusted for repeated test, the Ct value of the repeated result is still between 35 and 40, and the result is positive, otherwise, the result is negative; the detection sample is not amplified or Ct value is more than 40, and the detection sample is judged as negative.
Example 3 primer specificity and sensitivity verification
1. Primer specificity verification
1. The genome DNA and ddH of Bactrocera dorsalis (Rhagoletis cerasi), bactrocera zizanoides (Carpomya vesuviana), bactrocera dorsalis (Dacus (callantra)), bactrocera dorsalis (Bactrocera (Zeugodacus)), bactrocera dorsalis (Bactrocera (Bactrocera) dorsalais), bactrocera Medicata (Ceratitis (Ceratitis)) and Bactrocera guava (Bactrocera (Bactrocera) correcta), bactrocera vensis (Rhagoletis batava obseuriosa), bactrocera cusa (Bactrocera (Zeugodacus) tau), bactrocera Nalmeriensis (Anastrepha fraterculus), bactrocera dorsalis (Bactrocera (Zeugodacus)) and Bactrocera dorsalis 2 O is used as a template, and PCR reactions are respectively carried out by using specific primers OYF and OYR. The genomic DNA may be obtained by extracting DNA from a sample of adult fruit fly of the above-mentioned type.
2. PCR amplification
A. 25. Mu.L of PCR reaction system was used:
B. the PCR reaction tube is put into a common PCR instrument, and PCR amplification is completed according to the following reaction conditions:
the PCR reaction conditions were: pre-denaturation: 3min at 95 ℃; (denaturation: 95 ℃ C. For 15s; annealing: 65 ℃ C. For 1min; extension: 72 ℃ C. For 30 s) 30 cycles; extension: and at 72℃for 10min.
3. The results of primer specificity verification are shown in FIG. 1. In fig. 1, M: DL1200bp DNA Marker;1-6: bactrocera dorsalis; 7-8: fruit fly; 9-10: three-point bactrocera dorsalis; 11-12: melon fly; 13-14: fruit fly; 15-16: fruit fly in Mediterranean; 17-18: guava fruit fly; 19-20: sea buckthorn winds around the fruit fly; 21-22: pumpkin fruit fly; 23-24: fruit fly of south America; 25-26: fruit fly with stripe; 27-28: ddH 2 O。
From FIG. 1, it can be seen that only Bactrocera dorsalis can amplify clear bands, but no bands are amplified by other species of Bactrocera as well as by negative controls. The specificity verification result shows that the established PCR method for the Bactrocera dorsalis has high specificity, only a specific band with the fragment of 222bp appears in the reaction of the DNA template of the Bactrocera dorsalis, and no specific band appears in the same genus species, main Bactrocera dorsalis species of other genus and negative control.
2. Primer sensitivity verification
10-fold concentration gradient dilution was performed with the Europe Bactrocera dorsalis DNA template (86 ng/. Mu.L), respectively diluted 10 -1 -10 -8 And then, respectively taking the DNA stock solution and the diluent as templates, and detecting the sensitivity of the PCR reaction of the Bactrocera dorsalis according to the reaction system and the reaction conditions designed by the method.
FIG. 2 shows the results of primer sensitivity verification. In fig. 2, M: DL600bp DNA Marker;1: a stock solution; 2:10 -1 Dilution; 3:10 -2 Dilution; 4:10 -3 Dilution; 5:10 -4 Dilution;6：10 -5 dilution; 7:10 -6 Dilution; 8:10 -7 Dilution; 9:10 -8 Dilution; 10: ddH 2 O. From FIG. 2, it can be seen that the template DNA stock solution and 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Specific bands appeared in dilutions 10 -6 Dilution of amplified bands was relatively weak, dilution 10 -7 、10 -8 No specific bands appeared at all dilutions (fig. 2). It can be seen that 10 -5 The dilution is the lower limit of detection for this test. After conversion, the sensitivity of the PCR test of the Bactrocera dorsalis can reach 0.001 ng/. Mu.L, and the sensitivity is far better than that of the existing detection method.
Example 4
1. Primer specificity verification
1. The genome DNA and ddH of Bactrocera dorsalis (Rhagoletis cerasi), bactrocera zizanoides (Carpomya vesuviana), bactrocera dorsalis (Dacus (callantra)), bactrocera dorsalis (Bactrocera (Zeugodacus)), bactrocera dorsalis (Bactrocera (Bactrocera) dorsalais), bactrocera Medicata (Ceratitis (Ceratitis)) and Bactrocera guava (Bactrocera (Bactrocera) correcta), bactrocera vensis (Rhagoletis batava obseuriosa), bactrocera cusa (Bactrocera (Zeugodacus) tau), bactrocera Nalmeriensis (Anastrepha fraterculus), bactrocera dorsalis (Bactrocera (Zeugodacus)) and Bactrocera dorsalis 2 O is used as a template, and a specific primer and a probe OYF, OYR, OYP are respectively used for carrying out fluorescent quantitative PCR reaction.
2. Fluorescent quantitative PCR amplification
A. A 25. Mu.L fluorescent quantitative PCR reaction system was used:
| composition of the composition | Sample addition amount/. |
| 2×TaqMan real-time PCR master mix | 12.5 |
| 10 mu mol/L primer OYF | 0.5 |
| 10 mu mol/L primer OYR | 0.5 |
| 10 mu mol/L Probe OYP | 0.5 |
| 10-100 ng/. |
1 |
| ddH 2 O | Is added to 25 mu L |
B. The PCR reaction tube is put into a fluorescent quantitative PCR instrument, and PCR amplification is completed according to the following reaction conditions:
the fluorescent quantitative reaction conditions are as follows: 95 ℃ for 30s; collecting fluorescent signals after each cycle is finished in 95 ℃ for 5s and 65 ℃ for 35s and 40 cycles; 60 ℃ for 1min.
3. The specificity verification result is shown in fig. 3, and it can be seen from fig. 3 that 4 samples of the euonymus alatus DNA were co-amplified in fig. 3, each sample was set to 2 replicates, peaks were amplified, and none of the remaining bactrocera samples were amplified. The specificity verification result shows that the established fluorescence quantitative PCR method for the Bactrocera dorsalis has high specificity, and only the Bactrocera dorsalis DNA template can be amplified in the reaction, and CT is approximately equal to 18.5. The same genus species, other genus main bactrocera species and negative control are not amplified.
2. Primer sensitivity verification
10-fold concentration gradient dilution was performed with the Europe Bactrocera dorsalis DNA template (86 ng/. Mu.L), respectively diluted 10 -1 -10 -8 Thereafter, willThe DNA stock solution and the diluent are used as templates, and the sensitivity of the fluorescent quantitative PCR reaction of the Bactrocera dorsalis is detected according to the reaction system and the reaction conditions designed by the method.
In FIG. 4, A is stock solution, B is 10 -1 ,C:10 -2 ,D:10 -3 ,E:10 -4 ,F:10 -5 ,G:10 -6 ,H:10 -7 As can be seen from FIG. 4, the template DNA stock solution and 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 Amplification occurs in all dilutions, and CT is less than or equal to 35; dilution of 10 -6 、10 -7 Amplification occurs, but CT > 35;10 -8 Dilution was without amplification (fig. 4). It can be seen that 10 -5 The dilution is the lower limit of detection for this test. After conversion, the sensitivity of the PCR test of the Bactrocera dorsalis can reach 0.001 ng/. Mu.L, and the sensitivity is far better than that of the existing detection method.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> Manchurian customs technical center
<120> a specific primer, probe for identifying Bactrocera dorsalis and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gtaattgtta cagcccatgc c 21
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gtaaacagtt caacctgtc 19
<210> 3
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
aggagcacca gatatagctt ttcctcg 27
Claims (7)
1. A composition for identifying bactrocera dorsalis, comprising a primer and a probe; the sequence of the primer comprises: a sequence shown in SEQ ID NO.1 and a sequence shown in SEQ ID NO. 2; the sequence of the probe comprises a sequence shown in SEQ ID NO. 3.
2. The composition of claim 1, wherein the probe is further labeled with a fluorescent labeling group.
3. Use of a composition according to claim 1 or 2 for identifying bactrocera dorsalis.
4. A method for identifying bactrocera dorsalis, comprising the steps of: fluorescent quantitative PCR amplification of DNA of a sample to be identified using the composition of claim 1.
5. The method of claim 4, wherein the reaction conditions for fluorescent quantitative PCR amplification include: 30s at 95 ℃; collecting fluorescent signals after each cycle is completed in 5s at 95 ℃ and 35s at 65 ℃ for 40 cycles; 60 ℃ for 1min.
6. A kit for identifying bactrocera dorsalis, comprising the composition of claim 1.
7. The kit according to claim 6, further comprising one or a combination of several of a PCR amplification buffer, magnesium chloride, dNTPs, an enzyme for general PCR amplification, sterile water for fluorescent quantitative PCR amplification, a template DNA to be detected, a negative control, and a positive control.
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| CN202010924705.8A CN111979340B (en) | 2020-09-05 | 2020-09-05 | Specific primer and probe for identifying Bactrocera dorsalis and application of specific primer and probe |
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| CN101851675A (en) * | 2010-04-07 | 2010-10-06 | 中华人民共和国四川出入境检验检疫局 | Probe set of inspection and quarantine fruit fly and inspection method thereof |
| CN106048018A (en) * | 2016-06-08 | 2016-10-26 | 四川国际旅行卫生保健中心 | Method for detecting fruit flies of guava by using TaqMan probe, and primers and probe for detection |
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| CN105087786A (en) * | 2015-08-04 | 2015-11-25 | 福建出入境检验检疫局检验检疫技术中心 | Species specificity primer pair, kit and method for fast identifying Bactrocera cucurbitae |
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| CN101851675A (en) * | 2010-04-07 | 2010-10-06 | 中华人民共和国四川出入境检验检疫局 | Probe set of inspection and quarantine fruit fly and inspection method thereof |
| CN106048018A (en) * | 2016-06-08 | 2016-10-26 | 四川国际旅行卫生保健中心 | Method for detecting fruit flies of guava by using TaqMan probe, and primers and probe for detection |
Non-Patent Citations (1)
| Title |
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| From laboratory to point of entry: development and implementation of a loop-mediated isothermal amplification (LAMP)-based genetic identification system to prevent introduction of quarantine insect species;Simon Blaser 等;《Pest Management Science》;第74卷(第6期);第1504-1512页 * |
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