Schistosome infectious oncomelania detection kit and detection method thereof

A technology for detecting kits and schistosomiasis, which is applied in biochemical equipment and methods, microbiological measurement/testing, fluorescence/phosphorescence, etc. It can solve the problems of insufficient sensitivity and complex instruments and equipment, and achieve the effect of simple and fast operation

Inactive Publication Date: 2009-06-17
JIANGSU INST OF PARASITIC DISEASES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The purpose of the present invention is to provide a test kit and detection method for detection of schistosomiasis-infected oncomelania by loop-mediated isothermal DNA amplification, which can solve the shortcomings of previous schistosomiasis-infected oncomelania detection methods, such as insufficient sensitivity or the need for complex instruments and equipment. Identification and distribution investigation of schistosomiasis-infected snails in snail-bearing areas in schistosomiasis endemic areas

Method used

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  • Schistosome infectious oncomelania detection kit and detection method thereof
  • Schistosome infectious oncomelania detection kit and detection method thereof
  • Schistosome infectious oncomelania detection kit and detection method thereof

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Effect test

Embodiment 1

[0051] Example 1: Sensitivity Study

[0052] 1) Preparation of standard target DNA molecular template

[0053] Take the purified positive control sample DNA and dilute 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg, 0.1fg with TE buffer.

[0054] 2) Loop-mediated isothermal DNA amplification and result judgment

[0055] Take 10 0.2mL PCR thin-walled tubes, number them in sequence, and add the following components respectively: 2.5 μL of 10-fold LAMP buffer; 8 μL of dNTP (2mM); 2.5 μL of 10-fold primer mixture; Add 1 μL of DNA sample, and add 1 μL of deionized water to the No. 10 tube as the negative control; add 10 μL of deionized water to each tube. Mix well, boil in a water bath at 100°C for 5 minutes, put it on ice immediately, add 1 μL (8U) of Bst DNA polymerase, mix well, centrifuge briefly and place in a water bath at 60°C for 60 minutes; then transfer to a water bath at 80°C for 5 minutes. 5 μL of the amplification product was mixed with 2 μL of gel loading buffer con...

Embodiment 2

[0058] Example 2: Specificity studies

[0059] Genomic DNA of Schistosoma japonicum, Genomic DNA of liver fluke, Genomic DNA of Plasmodium, and Genomic DNA of Escherichia coli were respectively taken, amplified with the kit of the present invention, and sterilized deionized water was used as negative control at the same time. Only the schistosome genome showed a positive reaction, and the rest of the samples were negative. attached Figure 5 , 6.

Embodiment 3

[0060] Embodiment 3: Oncomelania sample detection on site

[0061] 1000 Oncomelania snails were collected on site in schistosomiasis endemic areas. Extract genomic DNA. Detection is carried out with the kit of the present invention, and at the same time, control is carried out by conventional PCR method detection. As a result, 1000 snails were detected with the loop-mediated isothermal DNA amplification kit of the present invention, and 56 snails were positive, with a positive rate of 5.6%. However, 52 snails were positive with a PCR method, and the positive rate was 5.2%. %, showing that the sensitivity of the loop-mediated isothermal DNA amplification method is higher than that of the conventional PCR method, and the operation is simpler.

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Abstract

A blood fluke infectious oncomelania detection kit and a detection method thereof belongs to the verminosis transmission medium detecting field. The invention provides a kit and a detection method for detecting blood fluke infectious oncomelania based on a loop-mediated isothermal DNA amplification technology (LAMP). According to the LAMP technology principle, six pairs of specific primers for amplifying a DNA fragment between the 20bp and 231bp of the schistosoma japonicum non-long terminal repeated inverse transcription transposon gene (AF412214) are designed. The LAMP method is constructed for detecting blood fluke gene in the body of the infectious oncomelania and the objective for distinguishing the blood fluke infectious oncomelania from the non-infectious oncomelania is reached. Comparing to the conventional blood fluke infectious oncomelania detection method the method of the invention has advantages of easy and fast operation, sensitive and specific without especial equipment and is suitable for bilharziasis controlling personnel use on site.

Description

technical field [0001] The invention relates to a kit and a detection method for detecting schistosomiasis infectious oncomelania based on loop-mediated isothermal DNA amplification, and belongs to the field of detection of parasitic disease transmission media. Background technique [0002] Oncomelania is the only intermediate host of schistosomiasis and the only transmission medium of schistosomiasis. Elimination of schistosomiasis-infected snails can block the spread and prevalence of schistosomiasis. To investigate the distribution of schistosomiasis-infected snails and selectively chemical molluscicide is an important means to improve the effect of blocking the transmission of schistosomiasis and to protect the ecological balance to the greatest extent. There are several methods for detecting schistosome-infected snails: [0003] Visual inspection [0004] Under natural light or artificial light, the color and light transmittance of the shell below the fourth spiral l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 余传信高琪殷旭仁
Owner JIANGSU INST OF PARASITIC DISEASES
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