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A PCR primer for identifying codling moth cell lines and its identification method

A technology of codling moth and cell line, applied in the field of agricultural biology, can solve the problems of poor repeatability, low sensitivity and the like, and achieve the effect of stabilizing the enzyme cleavage identification method

Inactive Publication Date: 2016-11-09
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current methods for identifying cell lines, such as cell line DNA amplification product fingerprinting (DAF), random amplified polymorphic DNA (RAPD), etc., the identification results are easily affected by many factors, and there are poor repeatability and sensitivity. low level problem

Method used

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  • A PCR primer for identifying codling moth cell lines and its identification method
  • A PCR primer for identifying codling moth cell lines and its identification method
  • A PCR primer for identifying codling moth cell lines and its identification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Amplification and sequence analysis of COI gene fragments in codling moth cell lines and several other insect cell lines

[0029] The applicant analyzed the COI gene partial sequence (SEQ ID No: 5) of the amplified codling moth cell line and the COI gene partial sequence (SEQ ID No: 6) amplified from the codling moth egg genome; carried out in the NCBI database Blast comparison, using DNASTAR software to compare and analyze the COI gene fragments of codling moth and several other insects, found that the COI gene nucleotide identity of codling moth and black beetle was 81.6%, and the COI gene of beet armyworm was 81.6%. The nucleotide identity of the gene is 87.9%, and it is 83.9% nucleotide identity with the COI gene of Trichoplusia spp. There are obvious differences in the sequences.

[0030] According to the sequence analysis results, the applicant designed the following primers:

[0031] Forward primer LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' (SEQ ID NO:...

Embodiment 2

[0049] The PCR-RFLP pattern of embodiment 2 several codling moth cell lines

[0050] (1) Extraction of genomic DNA from several codling moth cell lines

[0051] Seven codling moth cell lines Cp-E-4, Cp-E-6, Cp-E-9, Cp-E-10, Cp-E-11, Cp-E-12 and Cp - Genomic DNA was extracted from the cell suspension of E-13 with a MagExtractor-Genome kit to obtain genomic DNA solutions of each cell line of codling moth.

[0052] (2) PCR amplification of COI gene fragments in several codling moth cell lines

[0053] The genomic DNA solutions of several codling moth cell lines were respectively used as templates for PCR amplification to obtain PCR amplification products;

[0054] The PCR amplification system is:

[0055] Genomic DNA solution of codling moth cell line: 1 μL, 10 μmol / L forward primer: solution 1 μL, 10 μmol / L reverse primer solution: 1 μL, 5 U / μL Taq enzyme: 0.5 μL, 10×Taq buffer: 5 μL , 2.5mmol / L dNTP: 4μL, sterile pure water: 37.5μL.

[0056] The primer sequences are as fol...

Embodiment 3

[0066] Example 3 The difference between codling moth cell lines and other several insect cell lines PCR-RFLP patterns

[0067] (1) Extraction of genomic DNA of codling moth, black gill beetle, beet armyworm and Trichoplusia spp.

[0068] MagExtractor-Genome Reagent for Cell Suspensions of Codling Moth Cell Line Cp-E-10, Black Beetle Cell Line Hp-E-1, Beet Spodoptera Cell Line Se-1, and Trichoplusia Tn9-4s Genomic DNA was extracted using the cassette, and the genomic DNA solutions of the codling moth, black beetle, beet armyworm and Trichoplusiae cell lines were obtained.

[0069] (2) PCR amplification of COI gene fragments of codling moth, black gill beetle, beet armyworm and Trichoplusia spp.

[0070] The genomic DNA solution of codling moth and the genomic DNA solution of cell lines of beetle moth, beet armyworm and Trichoplusia spp. were respectively used as templates for PCR amplification to obtain PCR amplification products.

[0071] The PCR amplification system is:

...

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Abstract

The invention provides a PCR primer for identifying codling moth cell lines and an identification method thereof. The PCR primer pair for amplifying the mitochondrial COI gene fragments of codling moth and several other insect cell lines in the present invention has a forward primer sequence of SEQ ID NO: 1 and a reverse primer sequence of SEQ ID NO: 2. The optimized forward primer sequence is SEQ ID NO:3, and the reverse primer sequence is SEQ ID NO:4. The invention also provides a method for identifying the codling moth cell line. The present invention explores the differences in the mitochondrial COI gene fragment sequences between the codling moth cell line and several other insect cell lines from the molecular level, and establishes the identification of the codling moth cell line from the black beetle, beet armyworm, and Trichoplusia spp. method.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a PCR primer for identifying codling moth cell lines and an identification method thereof. Background technique [0002] Codling moth (Cydia pomonella) belongs to Lepidoptera, Lepidopteridae, native to south-central Europe. It invaded my country's Xinjiang in the 1950s, and now it has invaded Xinjiang, Gansu, Ningxia, Inner Mongolia, Heilongjiang and other regions, directly threatening the apple production areas in eastern my country, posing a serious threat to my country's apple industry, and has become an important quarantine pest in my country. . Studying the biological and physiological characteristics of codling moth is of great significance for the comprehensive management of the insect, but raising codling moth in non-endemic areas in China has certain difficulties and potential risks, and the establishment of codling moth cell lines will not cause cod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 李长友孟祥谦郑桂玲万方浩
Owner QINGDAO AGRI UNIV
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