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Tick nest type PCR (Polymerase Chain Reaction) specific primer and tick nest type PCR identification method

An identification method and specific technology, applied in the field of biology, can solve the problem of lack of molecular biological methods, and achieve the effect of shortening the required time.

Active Publication Date: 2017-05-31
温州出入境检验检疫局综合技术服务中心
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the identification of tick species in my country mainly relies on morphological methods, and there is no simple and stable molecular biological method.

Method used

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  • Tick nest type PCR (Polymerase Chain Reaction) specific primer and tick nest type PCR identification method
  • Tick nest type PCR (Polymerase Chain Reaction) specific primer and tick nest type PCR identification method
  • Tick nest type PCR (Polymerase Chain Reaction) specific primer and tick nest type PCR identification method

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Experimental program
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Effect test

Embodiment Construction

[0027] Design two pairs of specific primers based on the conserved region of tick 18s rRNA sequence

[0028] T18sF1:5`-GTAGTCATATGCTTGTCTC-3`;

[0029] T18sR1:5`-TCCGCAGGTTCACCTACGGA-3`;

[0030] T18sF2:5`-CAARTGTCTGCCYTATCAACT-3`;

[0031] T18sR2: 5`-CTTCCGTCAATTCCTTTAAG-3`.

[0032] Nested PCR specific steps

[0033] (1) Ticks

[0034] Ticks were intercepted from imported raw cowhide at Wenzhou Port, and preliminary morphological typing was carried out.

[0035] (2) Extract the tick genome

[0036] After morphological identification, one tick of each species was taken, and DNA was extracted from the genome according to the instructions of the genome extraction kit (ZYMOL), and the samples did not need to be treated with RNase.

[0037] (3) Cloning strategy

[0038] Cloning is done in two steps, according to figure 1 as shown,

[0039] ① Use tick genomic DNA as template (2 μl), T18sF1+T18sR1 as the first set of primers (1 μl each), LAtaq enzyme (0.25 μl), dNTP (2 μl)...

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Abstract

The invention provides a tick nest type PCR (Polymerase Chain Reaction) specific primer and a tick nest type PCR identification method. A more sensitive nest type PCR method is adopted; a single tick, even a trace genome DNA (Deoxyribonucleic Acid) extracted from partial tissue organs of the tick, can be used as a template and is used for identifying; somatotype and system evolution analysis on the tick is carried out on a gene level; in the nest type PCR identification method, a first round only needs 10 cycles and a second round needs 25 cycles, so that time needed by two turns of simple common PCR is shortened.

Description

technical field [0001] The invention specifically relates to the technical field of biology, in particular to a tick nest PCR specific primer and a tick nest PCR identification method. Background technique [0002] Ticks are obligate, non-permanent medical arthropods that parasitize terrestrial vertebrates and can cause harm to the host by directly biting and releasing toxins or carrying pathogens. In the investigation of tick species at entry and exit ports, the identification and evolutionary typing of ticks are of great significance. At present, the identification of tick species in my country mainly relies on morphological methods, and there is no simple and stable molecular biological method. Contents of the invention [0003] In order to solve the defects and deficiencies of the prior art, the present invention provides a tick-nest-type PCR-specific primer and a tick-nest-type PCR identification method, which can quickly and stably identify tick species at a low DNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6848C12Q1/6888C12Q2531/113C12Q2549/113C12Q2565/125
Inventor 王素华闫宝龙帅江冰吴绍强
Owner 温州出入境检验检疫局综合技术服务中心
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