A ring-mediated isothermal amplification and detection method of alligator nematode
A ring-mediated isothermal and Chinese alligator technology, applied in biochemical equipment and methods, microbe measurement/inspection, resistance to vector-borne diseases, etc., to achieve the effect of shortening the detection time
Active Publication Date: 2022-06-03
WANNAN MEDICAL COLLEGE
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[0004] In view of this, the purpose of the present invention is to propose a ring-mediated isothermal amplification and detection method of the Chinese alligator nematode, to solve all or part of the deficiencies in the prior art
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[0041] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
[0042] LAMP method ribonucleic acid amplification kit (DNA Amplification Kit), Japan Eiken Chemical Co., Ltd. (EIKEN CHEMICAL CO., LTD, Tochigi, Japan); LAMP reaction tube (ReactionTube), Japan Eiken Chemical Co., Ltd.; Visible dye (Te Visual Reagent, TVR), Tianjin Jieyite Biotechnology Co., Ltd.; plasmid pMD19-T vector and strain E.coli DH5α were purchased from Shanghai Sangon Biotechnology Co., Ltd. Tissue genomic DNA extraction kit (TIANampGenomic DNA Kit), Beijing Tiangen Biochemical Technology Co., Ltd.; stool genomic DNA extraction kit (TIANamp Genomic DNA Kit), Beijing Tiangen Biochemical Technology Co., Ltd.; Real-time turbidimeter La-320C, Japan Eiken Chemical Co., Ltd.; Spectrophotometer NanoDrop ND-1000, Thermo Fi...
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Abstract
The invention discloses a Chinese alligator nematode ring-mediated isothermal amplification and detection method, and relates to the technical field of parasite amplification detection. A LAMP reaction system is established according to the target sequence ITS of the nematode O. sinensis, and the nematode O. sinensis adult and infected Specific products were amplified in the feces of the Chinese alligator nematode, and the sensitivity of detecting adult genomic DNA was 3.46pg / μL, which was 10 times higher than that of the PCR method. This method does not require expensive instruments and only needs a water bath. Using the Bst DNA polymerase with strand displacement activity, the nucleic acid amplification reaction can be completed by incubating at 64°C for 1 hour. After the reaction, the nucleic acid dye can be added to observe the result, and there is no cross-reaction with other parasites, shortening the The on-site detection time is conducive to the promotion and use in grassroots health institutions, bedside detection and epidemiological investigations.
Description
technical field [0001] The invention relates to the technical field of parasite amplification detection, in particular to a ring-mediated isothermal amplification and detection method of Chinese alligator nematode. Background technique [0002] Diagnosis of parasitic diseases After undergoing general etiological and immunological diagnosis, with the continuous improvement of molecular biological diagnosis technology in recent years, nucleic acid detection methods that take target DNA sequences as detection objects have been able to accurately diagnose parasites. Diagnosis and testing. Among them, PCR technology has developed the fastest, and at the same time, PCR-based real-time quantitative PCR, multiplex PCR, nested PCR, etc. have developed this technology to a higher level. However, since the nucleic acid detection process often requires expensive equipment, professional and skilled personnel, complex operation processes, and lengthy test cycles, etc., it cannot meet the...
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IPC IPC(8): C12Q1/6844C12Q1/6888
CPCC12Q1/6844C12Q1/6888C12Q2531/119Y02A50/30
Inventor 赵金红谷生丽李媛媛
Owner WANNAN MEDICAL COLLEGE