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Specific primer and probe for identifying bactrocera humilis and application thereof

A specific and fruit fly technology, applied in the field of specific primers and probes for the identification of Bactrocera cerevisiae, can solve the problem that there is no national standard, industry standard, no method for Bactrocera cerevisiae, and cannot meet the needs of rapid, accurate and sensitive identification at daily ports work needs and other issues, to achieve the effect of short quarantine time, high sensitivity and high detection rate

Active Publication Date: 2020-11-24
满洲里海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process often takes 3-7 days to complete, which cannot meet the needs of rapid, accurate and sensitive identification at daily ports
[0006] At present, there is no method for rapid quarantine and identification of Bactrocera cerevisiae, and there is no corresponding national standard or industry standard

Method used

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  • Specific primer and probe for identifying bactrocera humilis and application thereof
  • Specific primer and probe for identifying bactrocera humilis and application thereof
  • Specific primer and probe for identifying bactrocera humilis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] This embodiment provides a group of specific primers for rapid identification of Bacterophae cerevisiae, and the primer sequences are as follows:

[0076] OYF: 5'-GTAATTGTTACAGCCCATGCC-3' (SEQ ID NO.1);

[0077] OYR: 5'-GTAAACAGTTCAACCTGTC-3' (SEQ ID NO. 2).

[0078] The present invention also provides a kind of PCR detection method for rapid quarantine identification of Bactrocera cerevisiae larvae, comprising the following steps in turn:

[0079] 1. Extraction of DNA from samples to be tested

[0080] Put the fruit fly sample into a 1.5ml centrifuge tube. The fruit fly sample can be adults, larvae or pupae, mash or add liquid nitrogen to grind, and use a commercial kit for DNA extraction.

[0081] 2. Ordinary PCR amplification

[0082] A. Use 25μL PCR reaction system:

[0083]

[0084] B. Put the PCR reaction tube into an ordinary PCR machine, and complete the PCR amplification according to the following reaction conditions:

[0085] The PCR reaction condition...

Embodiment 2

[0088] The present invention has also designed a group of fluorescent quantitative PCR primers and probes for fast identification of Bactrocera cerevisiae, and the sequences of primers and probes are as follows:

[0089] OYF: 5'-GTAATTGTTACAGCCCATGCC-3' (SEQ ID NO.1);

[0090]OYR: 5'-GTAAACAGTTCAACCTGTC-3' (SEQ ID NO.2);

[0091] OYP: 5'-FAM-AGGAGCACCAGATATAGCTTTTCCTCG-BHQ1-3' (the sequence of the probe is shown in SEQ ID NO.3).

[0092] The present invention also provides a fluorescent quantitative PCR detection method for rapid quarantine identification of Bactrocera cerevisiae larvae, comprising the following steps in turn:

[0093] 1. DNA extraction from samples to be tested

[0094] Put the fruit fly sample into a 1.5ml centrifuge tube. The fruit fly sample can be adults, larvae or pupae, mash or add liquid nitrogen to grind, and use a commercial kit for DNA extraction.

[0095] 2. Fluorescent quantitative PCR amplification

[0096] A. Use 25 μL fluorescent quantitati...

Embodiment 3

[0101] Example 3 Primer Specificity Verification and Sensitivity Verification

[0102] 1. Primer specificity verification

[0103] 1. Taking European cherry fruit fly (Rhagoletis cerasi), jujube fruit fly (Carpomya vesuviana), three-pointed fruit fly (Dacus (callantra) trimacula), melon fruit fly (Bactrocera (Zeugodacus) cucurbitae), fruit fly ( Bactrocera (Bactrocera) dorsalis), Mediterranean fruit fly (Ceratitis (Ceratitis) capitata), guava fruit fly (Bactrocera (Bactrocera) correcta), sea buckthorn fruit fly (Rhagoletis batava obseuriosa), squash fruit fly (Bactrocera (Zeugodacus) tau), Genomic DNA and ddH of Anastrephafraterculus and Bactrocera (Zeugodacus) scutellata 2 O is a template, and PCR reactions are carried out with specific primers OYF and OYR respectively. The above-mentioned genomic DNA can be obtained after DNA extraction from the above-mentioned species of fruit fly adult samples.

[0104] 2. PCR amplification

[0105] A. Use 25μL PCR reaction system:

...

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Abstract

The invention relates to a specific primer and a probe for identifying bactrocera humilis and application thereof. The sequence of the specific primer comprises a sequence shown in SEQ ID NO. 1 and asequence shown in SEQ ID NO. 2. The sequence of the probe comprises a sequence shown in SEQ ID NO. 3. A molecular biology quarantine identification method for quickly detecting the bactrocera humilis,which is established by adopting 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 [0001] The invention belongs to the technical field of insect quarantine and identification, and in particular relates to a specific primer, a probe and an application thereof for identifying Bactrocera cerevisiae. Background technique [0002] The European cherry fruit fly Rhagoletiscerasi (L.) belongs to Diptera Tephritidae, Tephritidae Tephritidae, and the genus Rhagoletis. Bacteria genus (non-Chinese species) is a quarantine pest that is prohibited from entering my country as stipulated in the "List of Imported Plant Quarantine Pests of the People's Republic of China". [0003] The European cherry fruit fly is mainly distributed in Europe and Asia. It is a highly destructive insect that seriously endangers the cherry industry in Europe and Asia. In Europe, if no chemical control is carried out, the infection rate can be as high as 100%, and the yield loss can be as high as 60%-100%. The fruit fly has a wide range of host plants, including various specie...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6851C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q1/686C12Q2531/113C12Q2563/107Y02E50/10Y02A50/30
Inventor 刘玮琦陈超姜帆李兰耿俊东郑敏刘振伟杨东来王旭
Owner 满洲里海关技术中心
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