Primers and method used for multi-target fast identification of bactrocera macquart

A fruit fly, multi-target technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., to achieve high sensitivity, high accuracy of identification results, and strong versatility

Pending Publication Date: 2018-12-21
CHINESE ACAD OF INSPECTION & QUARANTINE
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Problems solved by technology

[0007] In order to make up for the deficiency that the existing method can only quickly detect individual species of Drosophila, the present invention provides a multi-target rapid identification method of Drosophila, using the specific primers designed by the present invention to perform PCR detection on the genomic DNA of the sample to be tested , thus distinguishing Bactrocera from other economically important genera and closely related species such as Diptera

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  • Primers and method used for multi-target fast identification of bactrocera macquart
  • Primers and method used for multi-target fast identification of bactrocera macquart
  • Primers and method used for multi-target fast identification of bactrocera macquart

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Embodiment 1

[0050] Embodiment 1. Design of universal primers for Bactrocera species

[0051] The mitochondrial genome sequences of all Diptera insect species were retrieved in the GenBank database (the keyword used for retrieval was complete mitochondrial genome Diptera), and a total of 146 sequences were found, including the mitochondrial genome sequences of 16 species of Tephritidae insects. The mitochondrial genome sequence length of Diptera insects is about 20,000bp. DNAMAN software was used to perform multiple sequence alignments on the mitochondrial genome sequences of all Diptera insect species obtained, and the results showed that the mitochondrial genome sequences of Tephritidae species had an identity as high as 87.08% (e.g. Figure 3-5 ). In addition, the mitochondrial genome sequences of Anastrepha, Katrepha, Oligomanta, and Ceratifera were not published when the present invention was developed. Therefore, it is necessary to obtain the Specific primers for genus (Anastrephus...

Embodiment 2

[0056] Example 2. Establishment of a multi-target rapid identification system for Drosophila species

[0057] 1. DNA extraction

[0058] Utilize the blood / cell / tissue genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genomic DNA of 40 single-headed fruit fly samples (non-adult state or adult state) listed in Table 1 respectively, Refer to the manufacturer's instructions for specific operation procedures for subsequent primer verification.

[0059] 2. Verification of primer generality and specificity

[0060] The Drosophila species in Table 1 were used to verify the generality of the primer TBFdeg / TBR in Drosophila, and other Bactrocera species were used to verify the specificity of the primer TBFdeg / TBR to Drosophila.

[0061] Using genomic DNA as a template (with a concentration of 0.1-100 ng / μl), the designed primers are used for PCR amplification. The upstream primers are degenerate primers, and the downstream primers are spec...

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Abstract

The invention relates to the field of fruit fly quarantine technologies, in particular to primers and a method which are used for multi-target fast identification of bactrocera macquart. Nucleotide sequences of the specific primers used for multi-target fast identification of the bactrocera macquart are as shown in SEQ ID NO. 1 and SEQ ID NO. 2; the specific primers can separate the bactrocera macquart from anastrepha schiner, carpomya, ceratitis macleay, dacus, rhagoletis and other diptera species. The specific primers are used for identifying bactrocera macquart species, and have the advantages that the specificity is high, the sensibility is high, and the universality is high.

Description

technical field [0001] The invention relates to the fruit fly quarantine technology, in particular to a primer and a method for multi-target rapid identification of the fruit fly genus. Background technique [0002] The fruit fly belongs to the genus Bactrocera, which belongs to the Diptera Diptera tephritidae Tephritidae oligomane fruit fly subfamily Dacinae oligomane fruit fly family Dacini insects. Of the fruit fly species described in this genus, about 440 are related to fruit. Native to tropical Asia, Australia, and the South Pacific; a few species occur in warm temperate regions of Africa, Europe, and Asia; some species have been established in places such as Hawaii, French Guiana, and Suriname as a result of the modern fruit trade. Adults lay eggs on the fruit surface of fruits and vegetables to form holes, and larvae feed on the inside of the fruit, resulting in fruit rot or fruit drop. The damage can cause up to 100% economic loss, which not only constitutes a seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888
Inventor 姜帆田志清朱水芳张永江
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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