Method for detecting resistance of diamondback moth to Bt insecticidal protein Cry1Ac based on ABCC1 gene and kit thereof

A technology of insecticidal protein and detection method, which is applied in the field of bioengineering, can solve the problems of low sensitivity, long cycle time, high material requirements, etc., and achieve the effect of high sensitivity, accuracy and strong specificity

Inactive Publication Date: 2014-12-10
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of low sensitivity, long period, poor repeatability and high material requirements in the existing Plutella xylostella Bt insecticidal protein Cry1Ac resistance detection technology, the present invention intends to use specific fluorescent quantitative PCR primer screening and optimization of the reaction system and other steps , to establish a fast and accurate real-time fluorescent quantitative PCR molecular detection method and a fast, simple, accurate and efficient detection kit for detecting Bt resistance of Plutella xylostella, providing an effective tool for effective detection of Bt resistance in Plutella xylostella

Method used

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  • Method for detecting resistance of diamondback moth to Bt insecticidal protein Cry1Ac based on ABCC1 gene and kit thereof
  • Method for detecting resistance of diamondback moth to Bt insecticidal protein Cry1Ac based on ABCC1 gene and kit thereof
  • Method for detecting resistance of diamondback moth to Bt insecticidal protein Cry1Ac based on ABCC1 gene and kit thereof

Examples

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Effect test

Embodiment 1

[0047] Detection of midgut cDNA samples of 4th instar larvae of DBM1Ac-S and SZ-R by real-time fluorescent quantitative PCR technology and establishment of the detection kit and its application method.

[0048] 1. Taking the conserved region of the ABCC1 gene in the midgut of Plutella xylostella as the target sequence (SEQ ID NO.1), according to the principles of real-time fluorescent quantitative PCR primer design, the specific fluorescent quantitative primer sequence was designed as follows:

[0049] Forward primer (qCC1-F): 5′-GGTGGTGCTCATCTGCTACCTCAT-3′ (SEQ ID NO.2);

[0050] Reverse primer (qCC1-R): 5′-ATCCTGACACGCTCATCGGTTTT-3′ (SEQ ID NO.3);

[0051] The specific primer amplifies the fragment sequence as figure 1 As shown, its amplification specificity is as figure 2 (Among them, 1: the PCR amplified fragment of the target gene ABCC1; 2: the PCR amplified fragment of the internal reference gene L32; M: Marker I.) As shown in the figure, it can be seen from the figu...

Embodiment 2

[0087] The availability of the kit and detection method in Example 1 was verified by other three Bt-resistant diamondback moth populations DBM1Ac-R, NIL-R and SH-R. The specific experimental process is shown in Example 1.

[0088] The final fluorescent quantitative PCR reaction test results are as follows: Figure 6 Shown in, wherein, A: negative control sample of midgut cDNA of the 4th instar larvae of the Plutella xylostella population DBM1Ac-S sensitive to Bt insecticidal protein in A: in embodiment 1; positive control sample of midgut cDNA of 4th instar larvae of Plutella xylostella population SZ-R; C: cDNA test sample of midgut of 4th instar larvae of Plutella xylostella population SH-R resistant to Btk; D: insecticidal to Bt The midgut cDNA of the 4th instar larvae of the Plutella xylostella population DBM1Ac-R resistant to the protein Cry1Ac to be tested; E: the 4th instar larvae of the near isogenic line population NIL-R of the Plutella xylostella resistant to the Bt i...

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Abstract

The invention discloses a real-time fluorescent quantitative PCR method for detecting the resistance of a diamondback moth to Bt insecticidal protein Cry1Ac based on an ABCC1 gene and a kit thereof. The kit comprises the sequences of the following target gene specific primers: the sequence of a forward primer is shown in SEQIDNO. 2 and the sequence of a reverse primer is shown in SEQIDNO. 3. Since the method disclosed by the invention has the advantages of less manpower and samples, large detection amount, high sensitivity and strong specificity and is fast, convenient, accurate and reliable, the level of the resistance of the diamondback moth to the Bt insecticidal protein Cry1Ac can be effectively detected, the method is applicable to the early rapid diagnosis of the resistance of the diamondback moth to the Bt insecticidal protein Cry1Ac and can be also used for real-time monitoring and early warning/forecasting of the resistance of the diamondback moth in the field to the Bt insecticidal protein Cry1Ac and thus the method has broad application prospects.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a real-time fluorescent quantitative PCR detection method and a kit thereof for the resistance of diamondback moth to Bt insecticidal protein Cry1Ac. Background technique [0002] diamondback moth Plutella xylostella (L.), belonging to Lepidoptera (Lepidoptera) Plutellidae, is an important pest of cruciferous vegetables that is a worldwide hazard. In my country, since the 1970s, diamondback moth has gradually become the main pest of cruciferous vegetables in South China and the middle and lower reaches of the Yangtze River. At present, the harm of this insect has become increasingly serious in the vast North my country, Northeast China, and Northwest China. Plutella xylostella mainly damages leaves with larvae throughout the growth period of cruciferous vegetables, which greatly reduces the yield and quality of vegetables. When serious occurrences occur in individual fields, the y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2561/101
Inventor 张友军郭兆将康师朱勋吴青君王少丽徐宝云谢文
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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