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LAMP detection method for trichinella spiralises, special primers and kit thereof

A Trichinella and kit technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, etc., to achieve the effect of being suitable for large-scale popularization and application, simple operation, and efficient amplification

Active Publication Date: 2013-08-07
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there are no LAMP-specific primers and kits for detecting Trichinella spiralis on the market.

Method used

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  • LAMP detection method for trichinella spiralises, special primers and kit thereof
  • LAMP detection method for trichinella spiralises, special primers and kit thereof
  • LAMP detection method for trichinella spiralises, special primers and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Primer Design for LAMP Detection of Trichinella spiralis

[0039] The 1.6kb repetitive sequence of Trichinella spiralis (GenBank: X06625.1) was retrieved from the American Gene Database, and the homology analysis was performed by BLAST software. For the target DNA sequence, the primers for LAMP detection of Trichinella spiralis were designed with the software Primer design V4. As a result, three sets of primer pairs were optimized, F3 and B3 were the first set, FIP and BIP were the second set, LF and LB were the second set Three groups, the specific primer sequences are shown in Table 1. In the application, the three sets of primer pairs can be mixed and used in any proportion, and a specific optimized combination is given in the reaction system of Example 2 as an example.

[0040] Table 1 Primers used for LAMP detection of Trichinella spiralis

[0041]

Embodiment 2

[0042] Embodiment 2, establishment of the LAMP detection method of Trichinella spiralis of the present invention

[0043] The six primers used for LAMP detection of Trichinella spiralis obtained in Example 1 were used for LAMP detection of the Henan strain of Trichinella spiralis (from the Department of Human Parasites, School of Medicine, Zhengzhou University) to obtain the best reaction system and reaction conditions. The specific method is as follows :

[0044] 1. Determination of the best response system

[0045] Under the same reaction conditions (set at 63°C for 50 minutes), different concentrations of Mg were added to the reaction system 2+ , to determine the best reaction system, including the following steps:

[0046] 1) Using Trichinella spiralis genomic DNA (extract nucleic acid in the sample to be tested with the blood / cell / tissue genomic DNA extraction kit of TIANGEN company) as a template, carry out LAMP amplification under the guidance of the six primers obtai...

Embodiment 3

[0054] Embodiment 3, the specificity and sensitivity detection of the LAMP detection method of Trichinella spiralis of the present invention

[0055] One, the specific detection of the LAMP detection method of trichinella spiralis of the present invention

[0056] Trichinella spiralis (T1), native Trichinella spiralis (T2), pseudotrichinella spiralis (T4), Trichinella nersoni (T7), Ascaris lumbricus, pinworms, whipworms, Ancylostoma duodenale, S. Genomic DNA of Clochorchis sinensis, Fasciola brucei, Schistosoma japonicum, Taenia solium, Taenia saginata, and Taenia mansoni (all strains were obtained from the Department of Human Parasites, School of Medicine, Zhengzhou University) were used as templates, and double-distilled Water was used as a negative control to test the specificity of the best LAMP detection method for Trichinella spiralis obtained in Example 2.

[0057] The LAMP detection method of Trichinella spiralis of the present invention is to the specific turbidimete...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) detection method for trichinella spiralises, special primers and a kit thereof. The special primers used for LAMP detection of trichinella spiralises are designed according to the repeated sequences of a trichinella spiralis specific conservative gene sb4 shown in sequence 1 of the sequence table, and used for qualitative detection of trichinella spiralises in a sample to be detected. In conclusion, the method of the invention can detect trichinella spiralises under an isothermal condition, and has the advantages of rapidity, convenience, high efficiency, high specificity, high sensitivity and no need for complex equipment, thus providing a new technical platform for trichinella spiralis detection and being able to be used for trichinella spiralis screening and detection by animal husbandry production units, primary level medical and health care units, as well as all centers for disease control and prevention. Therefore, the method of the invention boasts wide market prospects as well as great economic and social benefits, and is suitable for large scope popularization and application.

Description

technical field [0001] The invention belongs to the molecular biology detection method of parasites in the field of biological technology, in particular to a LAMP detection method of Trichinella spiralis and its special primers and kit. Background technique [0002] Trichinella spiralis (Owen, 1835) Railliet, 1895] (Trichinella spiralis for short) is a food-borne zoonotic parasitic disease caused by Trichinella spiralis (Owen, 1835) Railliet, and is distributed in 66 countries or regions around the world. At present, the internationally recognized Trichinella genus is divided into 8 species and 3 genotypes, namely Trichinella spiralis (T1), native Trichinella spiralis (T2), Brunei (T3), pseudotrichinella spiralis (T4), Michaelis Trichinella spiralis (T5), Trichinella nelsoni (T7), Trichinella gentovii (T10) and Trichinella Zimbabwe (T11) and T6, T8, T9. Two species have been found in my country, namely Trichinella spiralis (T1) and native Trichinella spiralis (T2). [0003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 黄留玉袁静李雪莲刘威邹大阳王雪松王中全
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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