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Primer group capable of simultaneously detecting paragonimus westermani, paragonimus skriabini and paragonimus heterotremus and kit

A technology of primer sets and kits, applied in the field of primers and guards for the detection of Stein and Paragonimus heterogenus, can solve the problems of being unable to distinguish between past infection and current infection, prone to missed detection, etc., to achieve visualization, Easy operation and high sensitivity effect

Active Publication Date: 2020-02-14
ZHEJIANG ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Etiological diagnosis Examination of eggs with sputum or feces and biopsy of subcutaneous tissue, then in the incubation period or chronic infection, eggs are difficult to be observed, and missed detection is very easy; intradermal test or enzyme-linked immunosorbent assay (ELISA) Adsorption assays can be used for diagnostic testing, but cannot distinguish between past and current infections

Method used

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  • Primer group capable of simultaneously detecting paragonimus westermani, paragonimus skriabini and paragonimus heterotremus and kit
  • Primer group capable of simultaneously detecting paragonimus westermani, paragonimus skriabini and paragonimus heterotremus and kit
  • Primer group capable of simultaneously detecting paragonimus westermani, paragonimus skriabini and paragonimus heterotremus and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Real-time fluorescent LAMP verifies the effectiveness of primers

[0046] Add primers F3, B3, FIP and BIP, 8U Bst 2.0 DNA polymerase, 1.5 mM dNTP, 7.5 mM MgSO to the reaction tube 4 and ddH 2O, the concentrations of the outer primers F3 and B3 are both 0.2 μmol / μl, the concentrations of the inner primers FIP and BIP are both 1.6 μmol / μl, in addition, 1 μl of SYTO13 fluorescent dye with a concentration of 25 μM is added, and 2 μl of Weissler, Genomic DNA of adult worms of Steiner's and Paragonimus heterodiscoides was used as a template, and the genomic DNA was extracted by Tiangen Blood / Cell / Tissue Genomic DNA Extraction Kit (Product No. DP304). The reaction was performed on Bio-Rad's fluorescent quantitative PCR instrument. The specific reaction program was: react at 65°C for 1.5h and collect fluorescence data, and react at 95°C for 5min. Test results such as figure 1 As shown, the primers can simultaneously detect the adult genome DNA of Weissman, Steiner ...

Embodiment 2

[0047] Example 2 Nucleic acid detection test strip detects LAMP reaction product

[0048] Add the reacted LAMP product to the test strip, wait for 5-10 minutes, you can clearly see the T line and C line, the line is single, clear, and the background color is light, the test result of the sample is determined to be positive; the sample test In the test strip, if the T line cannot be clearly seen, but the C line appears clear red, it is judged that the test result of the sample is negative, and neither the T line nor the C line appears red, the test result is invalid. The results are described as figure 2 shown. The experimental results are as image 3 As shown, the test strips of the LAMP reaction products whose templates are the genomic DNAs of adults of Weiss, Steiner and Paragonimus heterodiscoides are also positive, while the templates are the genomic DNAs of Trichinella spiralis, Anisakis, Schistosoma, and Angiostrongylus cantonensis Or the absence of bands in distille...

Embodiment 3

[0049] The detection result of embodiment 3 kit to clinical sample

[0050] This kit was used to test 124 canine blood samples. Genomic DNA was extracted from the blood samples to be tested, and 5 μl was added to the LAMP reaction solution as a template, and reacted in a water bath at 63°C for 45 minutes, and the reaction product was added to the test paper. On the bar, observe the result after 5-10 minutes. The results showed that 9 positive samples were detected with a positive rate of 7.25%, and 115 negative samples were detected with a negative rate of 92.75%. The results are shown in Table 1.

[0051] Table 1 test strip clinical test results

[0052] positive(%) feminine(%) The detection rate(%) 9(7.25) 115(92.75)

[0053] Result Judgment: In the test strip for sample detection, if T line and C line can be clearly seen, the line is single, clear, and the background color is light, then the test result of the sample is determined to be positive f...

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Abstract

The invention discloses a primer group capable of simultaneously detecting paragonimus westermani, paragonimus skriabini and paragonimus heterotremus, a kit and a method. The kit comprises a test paper strip, a paragonimus DNA positive control solution and a detection solution, wherein the detection solution specifically comprises an LAMP buffer solution, the primer group, a Bst 2.0 DNA polymerase, dNTP, MgSO4 and ddH2O, and the primer group comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP and a probe Probe. The kit can be used for rapidly detecting genomic DNA of paragonimus in a sample. The invention provides the method for detecting the genomic DNA of the paragonimus. The method can be used for detecting genomic DNA of adults, encysted cercariaand ova of the paragonimus. The kit is high in sensitivity, high in specificity, relatively good in stability and simple in operation, can better meet the requirements of people of different hierarchies and facilitates popularization to grass roots.

Description

technical field [0001] The invention belongs to the diagnostic technique in the field of biotechnology, and in particular relates to primers for detecting Weiss, Stein and Paragonimus heterodiscoides and their application. Background technique [0002] The adult worms of the genus Paragonimus live in the lungs of the host, commonly known as lung flukes. So far, nearly 50 species of Paragonimus have been reported in the world, of which 32 species have been reported in my country, and Paragonimus wescheri, Paragonimus stinkii and Paragonimus heterodiscoides are the most prevalent, and all of them can parasitize in human body. Adults of Paragonimus often parasitize the lungs, but their juveniles sometimes parasitize tissues and organs such as the liver, brain, eye sockets, and subcutaneous tissue, causing damage to various tissues and organs due to the migration of parasites in the body. Because the symptoms of paragonimiasis are complex and often involve multiple organs, the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2565/625
Inventor 卓洵辉孔庆明童群波陆绍红丁豪杰郑斌楼涤丁建祖陈睿
Owner ZHEJIANG ACAD OF MEDICAL SCI
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