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Carmine spider mite SCAR (sequence characterized amplified region) mark and specificity PCR (polymerase chain reaction) detection method thereof

A technology of Tetranychus cinnabarinus and detection method, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., and can solve the problems of inability to conduct accurate analysis, time-consuming, labor-intensive, and long-term consumption

Inactive Publication Date: 2011-09-14
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technique also has some shortcomings and limitations. For example, the sequences of some spider mite species have not been registered in GenBank, so some species cannot be effectively clustered, so accurate analysis cannot be performed; Sequencing after PCR amplification will consume a lot of time and financial resources
Techniques such as restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphisms (Single-Stranded Confirmation Polymorphisms, SSCP) are also commonly used for molecular identification of insect species, but the process Complicated, requiring enzyme digestion, amplification or PCR amplification, polyacrylamide gel electrophoresis combined with silver staining methods, etc., all of which take a long time and are time-consuming and laborious

Method used

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  • Carmine spider mite SCAR (sequence characterized amplified region) mark and specificity PCR (polymerase chain reaction) detection method thereof
  • Carmine spider mite SCAR (sequence characterized amplified region) mark and specificity PCR (polymerase chain reaction) detection method thereof
  • Carmine spider mite SCAR (sequence characterized amplified region) mark and specificity PCR (polymerase chain reaction) detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Cloning of specific fragments of Tetranychus cinnabarinus

[0022] 1. Materials

[0023] 1.1 Tetranychus tested

[0024] Tetranychus cinnabarinus was provided by the School of Plant Protection, Nanjing Agricultural University. The sympatric control species Tetranychus urticae and Tetranychus truncation were provided by Nanjing Agricultural University and the Plant Protection Institute of Fujian Academy of Agricultural Sciences respectively. These three species were detected by traditional microscope The morphological characteristics of the male external genitalia and the color of the female adult mites are observed to identify them.

[0025] Hawthorn Tetranychus was used as the control species of allopatric spider mites, which was provided by the Fruit Tree Institute of Shandong Academy of Agricultural Sciences.

[0026] The above-mentioned spider mites are all known spider mites, and all of them are preserved in our laboratory and can be released to the pub...

Embodiment 2

[0042] Example 2 Designing primers based on Tetranychus cinnabarinus specific fragments and obtaining SCAR markers

[0043] Based on Seq ID No.1, design a pair of specific primers:

[0044] Cinnabar-230F(5’-CGG AAA TCA GAC TAT GTG-3’)

[0045] Cinnabar-230R (5’-CTG ​​TTG ATA ACG GAG AAA-3’),

[0046] The reaction system was set at 25 μl, of which 2×ES Taq Master Mix (including ES TaqE 5U / μl) was 12.5 μl, 230F / 230R was 1 μl, and DNA template was 1.25 μl.

[0047] The reaction program was as follows: pre-denaturation at 94°C for 3 min, followed by 30 cycles (denaturation at 94°C for 1 min, annealing at 57°C for 45 sec, extension at 72°C for 1 min), and finally extension at 72°C for 10 min. The expected amplified fragment size is 230bp.

[0048] The specific SCAR primer pair: Cinnabar-230F (5'-CGG AAA TCA GAC TAT GTG-3') and Cinnabar-230R (5'-CTG TTG ATA ACG GAG AAA-3') was used to control the indoor preservation of Tetranychus cinnabarinus Populations were amplified by PCR, ...

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Abstract

The invention relates to a carmine spider mite SCAR (sequence characterized amplified region) mark and a specificity PCR (polymerase chain reaction) detection method thereof, belonging to the field of plant insect identification technologies. The carmine spider mite SCAR mark has the nucleotide sequence as shown in Seq (sequence) ID (identity) No.2; and the specificity PCR detection method of carmine spider mite is characterized in that specificity forward and reverse primers adopted in a PCR amplification system are designed by the nucleotide sequence of the SCAR mark described in claim 1, and an amplification region of the specificity forward and reverse primers is arranged on the nucleotide sequence of the SCAR mark. Compared with the traditional identification method, the method is simple, fast, time-saving, labor-saving and easy to master for staff in a classification major and the non-classification majors, thereby being the molecular biological method which can fast identify the carmine spider mite.

Description

technical field [0001] The invention relates to plant insect identification technology, in particular to a Tetranychus cinnabarinus SCAR marker and a specific PCR detection method thereof. Background technique [0002] Tetranychus cinnabarinidae belongs to Arachnidae, Acariidae, Tetranychidae, commonly known as spider mites, and is an important pest in agricultural production. It has a wide range of hosts and mainly damages vegetables, cotton, wheat and other important crops. Tetranychus cinnabarinus causes adult mites and nymphs to gather on the back of plant leaves to pierce and suck, causing chlorotic spots to appear on the leaves. In severe cases, it will appear burnt, the leaves will dry up and fall off, and even the plants will die in pieces. [0003] The female adult mite of Tetranychus cinnabarinus is pear-shaped, 0.5mm in size, reddish-brown or rust-red in color; the male adult mite is about 0.3mm in size, and the end of the abdomen is slightly pointed. Such tiny i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王少丽戴宇婷王相晶张友军吴青君徐宝云
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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