Fluorescent quantitative PCR reagent kit for fast detecting Japanese blood fluke

A fluorescent quantification and schistosomiasis technology, which is applied in measuring devices, microbial determination/inspection, and material analysis through optical means, can solve problems such as inaccurate quantification, false positives, and low sensitivity

Inactive Publication Date: 2008-02-27
王业富
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In terms of clinical detection, traditional etiological methods and serological methods have disadvantages such as low sensitivity, poor specificity, false positives, time-consuming and labor-intensive, etc.; although conventional PCR method has the advantages of simplicity, rapidity and sensitivity, it cannot be accurately quantified and post-PCR Deal with problems such as false positives caused by pollution; therefore, it is necessary to develop an accurate, sensitive, fast, and pollution-free clinical testing method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: kit composition and preparation

[0045] ① DNA extraction reagent (lysate)

[0046] Prepare for yourself, lysate: 200mmol / L NaCl, 100mmol / L Tris-HCl, pH 8.5, volume fraction 1% SDS, 50mmmol / L EDTA pH 8.5, 3mg / ml proteinase K (add just before use).

[0047] ② Fluorescence PCR 10×Buffer composition:

[0048] 500mM KCl, 100mM Tris-HCl (PH9.025°C);

[0049] ③Fluorescence quantitative PCR reaction solution: PCR 10×buffer 5μl, forward primer and reverse primer 2μl (10μmol / L), fluorescent probe 1μl (10μmol / L), MgCl 2 7μl (25mmol / L), dNTPs 1μl (10mmol / L), HOTSTART Taq DNA polymerase 0.4μl (5U / μl), sterile double distilled water 30.6μl. Fluorescent probes were used at a concentration of 10 μmol / L.

[0050] ④Standard positive template stock solution: the concentration is 10 8 Copy / μl standard positive template PU-SJ.

[0051] ⑤ Negative quality control standard: sterile double distilled water

Embodiment 2

[0052] Example 2: Rapid detection of Schistosoma japonicum using a kit

[0053] ① Add 0.5ml of DNA extraction solution to the specimen and mix well, bathe in 65°C water for 30min and shake gently from time to time, centrifuge at 10000g for 5min, take the supernatant and add 2μl RNaseA at 37°C for 30min, wash with phenolform (phenol: chloroform: isoamyl alcohol) =25:24:1) extracted twice, washed with cold ethanol, dissolved in 100 μl sterile double distilled water, and 1 μl was used for PCR reaction.

[0054] ② Serially dilute the positive standard template (reagent d) to 10 7 copies / μl, 10 6 copies / μl, 10 5 copies / μl, 10 4 copies / μl, 10 3 copies / μl.

[0055] ③Respectively take 49 μl of fluorescent quantitative PCR reaction solution (reagent c), take 1 μl each of SJ DNA obtained in step a) and SJ positive standard template diluted in step b), and set up a negative control, and add them to different PCR reaction tubes respectively. Perform PCR detection in parallel on a fl...

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Abstract

The invention discloses a fluorescence quantitative PCR reagent box for rapid detection of Schistosoma japonicum, relates to gene detection technology of a kind of pathogen of zoonotic infectious disease, suitable for qualitative and quantitative detection of Schistosoma japonicum. The invention comprises of DNA extracting solution, fluorescence quantitative PCR reaction solution, standard positive template PU-SJ and negative quality control standard sample; the fluorescence quantitative PCR reaction solution contains specific primers and fluorescence probe of Schistosoma japonicum. The invention was accurate in quantitative detection, quick with good specificity and high sensitivity; can identify and detect Schistosoma japonicum and Chinese schistosomiasis; the steps are simple with good repeatability; can qualitatively and quantitatively detect the DNA samples of all three species of Schistosoma japonicum group, can replace traditional aetiology methods, oncomelania crushing method of detecting the snail of schistosome cercariae and larva escape method.

Description

technical field [0001] The invention relates to a gene detection technology for zoonotic infectious disease pathogens, in particular to a fluorescent quantitative PCR (polymerase chain reaction) kit for rapid detection of Schistosoma japonicum, which is suitable for qualitative and quantitative detection of Schistosoma japonicum. Background technique [0002] Schistosoma is also called Schistosoma. In 1851, the German pathologist Theodor Bilharz first discovered Schistosoma haematobium adult worms when he performed an autopsy on a hematuria patient in a Cairo hospital. So far, 19 species of Schistosoma have been found. Six of the 19 species of schistosomes parasitize humans: Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma mekongi and Schistosoma malayi (Wu Zhengjian, Chinese Medical Encyclopedia of Parasitology and Parasitology〔M ], Shanghai Science and Technology Press, 1984.). The 19 species of schistosomes of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/00C12Q1/68
Inventor 王业富周立赵友云
Owner 王业富
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