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Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method

A ring-mediated isothermal and Plasmodium technology, which is applied in the fields of biochemical equipment and methods, resistance to vector-borne diseases, and measurement/testing of microorganisms, can solve the problems of general screening of Plasmodium, poor specificity, and small size of Plasmodium. And other issues

Inactive Publication Date: 2011-04-13
中华人民共和国徐州出入境检验检疫局
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Problems solved by technology

Due to the small size and similar shape of Plasmodium, it is difficult to stain and detect, and only experienced personnel can make a correct diagnosis; at the same time, microscopic examination is time-consuming and laborious, which is not suitable for the detection of large-scale samples and is not conducive to the general screening of the above-mentioned Plasmodium.
(2) Polymerase chain reaction (PCR), which has poor specificity and has the advantage that the sensitivity can reach 98% to 100%. However, it requires PCR amplifiers, gel electrophoresis and other related expensive equipment, which is not conducive to primary medical epidemic prevention Institutional and on-site rapid determination

Method used

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  • Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method
  • Loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method

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Embodiment

[0043] (1) Nucleic acid extraction: Take 1ml of the bacterial suspension cultured on a shaker at 37°C for 10 hours, centrifuge at 10,000rpm for 5min, discard the supernatant, add 100μl of sterile water and shake well (or store it in a refrigerator at 4°C). Pick 2-3 colonies on the plate and put them in 100 μl sterile water). Boil at 100°C for 10 minutes, centrifuge at 10,000 rpm for 5 minutes in a desktop centrifuge, and the supernatant is genomic DNA, which can be directly used as a template for nucleic acid amplification reactions.

[0044] (2) LAMP reaction: Prepare the above reaction system, mix the reaction system evenly, and distribute it into 0.2ml PCR reaction tubes, 19ul per tube, add the template extracted in step (1) to each PCR reaction tube, 1ul per tube , 1ul each of the positive control substance and the negative control substance were used as the positive control and the negative control respectively, and mixed evenly. Put it in a constant temper...

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Abstract

The invention discloses a set of loop-mediated isothermal amplification technology-based plasmodium genus and species nucleic acid screening method and belongs to the field of biological detection. The method is performed by loop-mediated isothermal amplification (LAMP) technology, specific positions of target genes are amplified by using an LAMP technology platform through specific primers of plasmodium genera and specificity primers of plasmodium species, the plasmodium genera are screened or detected at a molecular level under assistance of positive and negative quality control and an internal control detection system, and plasmodium species screening or detection is performed on four kinds of plasmodia, namely Plasmodiumfalciparum, Plasmodiummalariae, Plasmodiumovale and Plasmodiumvivax which can make humans infected with malaria in plasmodium genus organisms. The invention has the characteristics that: the method is simple, economic and rapid, and has high sensitivity, high specificity and wide application prospect.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a nucleic acid screening method for Plasmodium genus and species based on loop-mediated isothermal amplification (LAMP) technology; containing Plasmodium falciparum ( Plasmodium falciparum ), Plasmodium malariae ( Plasmodium malariae ), Plasmodium ovale ( Plasmodium ovale ) and Plasmodium vivax ( Plasmodium vivax )) Nucleic acid screening method. Background technique [0002] At present, the detection methods of Plasmodium genus and species are mainly microbial culture identification and multiplex PCR detection technology. (1) Microscopic examination, microscopic examination of thick blood film and thin blood film is still the gold standard for detection of malaria parasites. However, there are some problems in microscopic examination as a detection method. Due to the small size and similar shape of Plasmodium, it is difficult to stain and detect, and only experienced ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 易海华房超吴萍兰宋阳威徐波孙慧宇王伟邵景东孙立新钱进杨志俊
Owner 中华人民共和国徐州出入境检验检疫局
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