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Kit for detecting fasciola hepatica by using PCR specificity

A fasciola and kit technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of easy misdiagnosis and missed diagnosis, low detection rate, limited sensitivity, etc., and achieve result judgment. Objective, method-specific, and sensitive effects

Inactive Publication Date: 2011-02-02
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a kind of reagent kit that adopts PCR specificity to detect Fasciola, and described this kit that adopts PCR specificity to detect Fasciola will solve the detection rate of the method for detecting Fasciola in the prior art. High, many false positives, time-consuming and labor-intensive, prone to misdiagnosis and missed diagnosis, technical problems with limited sensitivity

Method used

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  • Kit for detecting fasciola hepatica by using PCR specificity
  • Kit for detecting fasciola hepatica by using PCR specificity
  • Kit for detecting fasciola hepatica by using PCR specificity

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Embodiment 1

[0044] The present invention is a kit for detecting Fasciola by specific PCR, which contains DNA lysate, PCR reaction solution and Fasciola hepatica, Fasciola large, and intermedium DNA positive control solution, and the DNA lysate is composed of cell lysate , EDTA, proteinase K and RNase A solution, in the DNA lysate, the final concentration of the cell lysate is 7-10uM, the final concentration of EDTA in the DNA lysate The concentration is 0.5M, the final concentration of the proteinase K in the DNA lysate is 18-20mM, the final concentration of the RNase A in the DNA lysate is 3-5uM, the In the PCR reaction solution, dATP, dTTP, dGTP, dCTP, downstream primers for amplifying Fasciola hepatica, downstream primers for amplifying Fasciola large, shared upstream primers for amplifying Fasciola hepatica and Fasciola large, and MgCl 2 The sequence of the downstream primer for amplifying Fasciola hepatica is 5'-CCAATGACAAAGTGACAGCG-3', and the downstream primer for amplifying Fascio...

Embodiment 2

[0049] A preferred test kit of embodiment 2

[0050] The kit contains DNA lysate, which contains 100ml of a mixed solution of cell lysate (7μM), pH 8.0 (final concentration 0.5M) EDTA, proteinase K (final concentration 20mM) and RNase A solution (final concentration 5μM); 100 reactions of PCR reaction solution (25 μL / reaction): dATP, dTTP, dGTP, dCTP with a final concentration of 200 μM, primers for amplifying Fasciola hepatica with a final concentration of 50 pmol / μL, and amplification with a final concentration of 50 pmol / μL Downstream primers of Fasciola grandis, common upstream primers for amplifying Fasciola hepatica and Fasciola grandis at a final concentration of 50 pmol / μL, MgCl at a final concentration of 2 mM 2 Mixed solution, 25 μL Taq DNA polymerase (6U / μL); Fasciola hepatica, Fasciola large, and Fluke intermedius DNA positive control solution.

[0051] Embodiment 2 kit specificity test

[0052] Use 1 μL each of the control sample DNA of Fasciola hepatica, Fascio...

Embodiment 3

[0063] The sensitivity test of embodiment 3 kits

[0064] Firstly, the DNA of the adult Fasciola fluke was extracted, diluted, vortexed and mixed, and the total DNA content was detected according to the operating procedures of the Eppendorf Biophotometer Nucleic Acid Protein Analyzer. Fasciola hepatica was 44.16ng, Fasciola large was 140.96ng. The DNA was diluted 25, 50, 100, 200, 400, and 800 times, and the PCR amplification conditions were the same as above, and a blank control was set at the same time. PCR products were detected by 1.0% agarose gel electrophoresis to determine their sensitivity. The test results show that the PCR detection method has high sensitivity, and can detect 0.11ng of Fasciola hepatica and 0.35ng of Fasciola large ( image 3 and Figure 4 ).

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Abstract

The invention discloses a kit for detecting fasciola hepatica by using PCR specificity. The kit comprises DNA cracking solution, PCR reaction solution and fasciola hepatica, fasciola gigantica and middle fluke DNA positive contrast solution. The invention also provides a method for detecting the fasciola hepatica by using a specific PCR method. The method can rapidly, specifically and flexibly diagnose and detect fasciola hepatica cercaria in snails and fasciola hepatica ova in excrements of infected animals. A rapid, specific and sensitive PCR method is established for detecting the imagoes, the cercaria and the ova of the fasciola hepatica, the fasciola gigantica and the middle fluke, so that the imagoes, the cercaria and the ova of the fasciola hepatica, the fasciola gigantica and the middle fluke can be accurately differentiated. The kit of the invention has the advantage of simple and programmed operation, and the method has the advantages of high specificity, high sensitivity and objective result judgment and can be used for rapidly differentiating the fasciola hepatica.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a kit, in particular to the specific PCR detection of Fasciola hepatica, Fasciola large, intermediate trematode adults, cercariae and eggs, specifically a method for specific detection of Fasciola by PCR kit. Background technique [0002] Fascioliasis is a helminth disease caused by parasitizing the liver and bile ducts of domestic animals by flukes of the genus Fasciola (Fasciolidae, Fasciolidae, and Intermedium) in the Fasciolidae family. It mainly occurs in ruminants (cattle, buffalo, sheep, goat, camel, etc.), and the clinical symptoms are mainly chronic emaciation and exhaustion caused by nutritional disorders and poisoning; the pathological features are chronic cholangitis and hepatitis. Humans can also be infected with fasciola. The main pathogens of this disease are Fasciola hepatica and Fasciola large. At present, reports of intermediate trematode infecting animals ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 陈家旭艾琳陈木新陈韶红李鑫朱兴全
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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