PCR detection method for quickly detecting large coptotermes formosunus

A detection method, termite technology, applied in the field of PCR detection, can solve the problems of low reliability, poor accuracy, low probability of winged adults, etc., to achieve the effect of ensuring public health safety, protecting production safety, and maintaining ecological balance

Inactive Publication Date: 2010-03-10
中华人民共和国珠海出入境检验检疫局
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Problems solved by technology

Therefore, the traditional morphological identification is not only subjective but also cumbersome. It is difficult to reliably analyze and identify the differences between similar species, and it is even more difficult to reveal the genetic relationship of different groups of insects in the evolution process.
The difficulty of identifying the species of the genus Coptotermes is recognized by the world's termite taxonomy experts. The various species of this genus are very similar to each other in shape. At the same time, a considerable number of species have variations in the head shape of the soldier ant, and a large number of measures are used to measure the size of each part of the termite. As a feature to distinguish similar species, it is sometimes an important feature, which also brings great difficulties to the identification work
In addition, it is not easy to obtain the rank of soldier ants during the on-site quarantine of termites, and the probability of obtaining winged adults is even lower. It is difficult to identify the species level based on the morphological characteristics of worker ants, and the results are not reliable.
This traditional identification method based on the main characteristics of soldier ant morphology and anatomy, combined with morphological measurement values, is greatly affected by environmental and geographical conditions, and termites with different geographical distributions have different morphological data measurements. All consistent, resulting in low reliability and poor accuracy in the actual identification process, missed inspections, wrong inspections are common, and do not meet the requirements of rapid customs clearance at inspection and quarantine ports

Method used

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  • PCR detection method for quickly detecting large coptotermes formosunus

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Embodiment 1

[0040] This embodiment designs the specific primers of our termites. In the experiment, we use 6 kinds of termites to carry out PCR specificity to our termites, Coptotermes formosanus, Coptotermes saipang, Coptotermes indo-Burmese, Coptotermes termites and Coptotermes japonica. For testing, the sample numbers and sources are shown in the table below:

[0041]

[0042]

[0043] like figure 2 As shown, the designation of the routine PCR upstream primer number is CC-F5, located at the 407bp-427bp position of our termite ITS1, see sequence number NO.1; the downstream primer number is CC-R5, located at the 641bp-661bp position of our termite ITS2 For the position, see SEQ ID NO: 2, the size of the amplified fragment is 254bp.

[0044] The specific detection steps are:

[0045] For the preparation of DNA templates, a termite was taken, rinsed in double distilled water, blotted dry with absorbent paper, and its body surface attachments and abdominal contents were removed und...

Embodiment 2

[0050] This embodiment adopts real-time fluorescent PCR detection, (such as figure 2 Shown) fluorescent primers and probes are designed to be located at ITS2, the upstream and downstream primers and probes are numbered CC-F6, CC-R8 and CC-TZ-6 respectively, and the sequence numbers in the sequence list are NO.3 and NO. 4 and NO.5, the size of the predicted amplified fragment is 155bp, the upstream primer CC-F6 sequence is 5'-AGGCCGCGGGCCCGAAGATGG-3', the downstream primer CC-R8 sequence is 5'-CCGAGATCTCTCTCGTATTTTC-3', the primer probe CC- The sequence of TZ-6 is 5'-FAM-ACGACAGCACTGTCTTAATCTTCG-TAMRA-3'.

[0051] The basic detection steps are the same as in Example 1, the difference lies in the establishment of the PCR reaction system: the total volume is 20 μl, and the components are as follows: 2 μl of 10×PCR buffer, 2 μl of MgCl 2 (25mmol / mL), 1.6μl dNTP mixture (10mmol / mL each), 0.5μl each of upstream and downstream primers (CC-F6, CC-R8, 10μmol / mL), CC-TZ-6 probe (10μmo...

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Abstract

The invention discloses a PCR detection method for quickly detecting large coptotermes formosunus, which comprises conventional PCR detection and real-time fluorescence PCR detection. The PCR detection method comprises the following steps: preparing a DNA template for a sample to be detected, designing a specific primer and/or a probe, establishing a PCR reaction system for PCR amplification, andobserving, recording and photographing on a gel imaging system. With the advantage of high speed, stability, reliability and sensitivity, the PCR detection method for quickly detecting large coptotermes formosunus is applied to the detection of different-grade samples of large coptotermes formosunus, and lays a foundation for China's coptotermes formosunus class in molecular classification and therapid identification of sibling species. The result of study can be directly applied to the quarantine identification of large coptotermes formosunus in inward and outward plant quarantine, and has important significance to the production safety protection of agriculture and forestry, biological balance maintenance, public health safety insurance in China.

Description

technical field [0001] The invention relates to the field of molecular diagnosis of biological species, in particular to a PCR detection method for rapid detection of termites. Background technique [0002] Termites belong to the order Isoptera of the order Insecta, and there are more than 2860 species in 7 families, 286 genera, and they are a class of worldwide important pests. There are more than 400 kinds of termites recorded in my country, and there are many kinds of termites with great harm, especially the genus Coptotermes Wasmann (1896). One of the most threatening termite species to spread and spread. This termite has strong adaptability, wide distribution, and rapid reproduction. It can not only harm a variety of trees, eat hard wood and even cellulose-containing wood processed products such as paper, cotton cloth, leather products, etc., but also harm the plastic protection of buried communication cables. Nesting and agroforestry crops. The danger of this termite...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 乐海洋张卫东廖力
Owner 中华人民共和国珠海出入境检验检疫局
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