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Fluorescent quantitative PCR (FQ-PCR) kit for rapidly detecting Toxoplasma gondii

A fluorescence quantification and Toxoplasma gondii technology, which is applied in the direction of fluorescence/phosphorescence, microbe-based methods, microbiological measurement/inspection, etc., can solve the problems of long culture time, PCR pollution, and limited diagnostic value, etc. The effect of good repeatability and fast detection speed

Inactive Publication Date: 2009-12-30
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In terms of clinical detection, the traditional etiological detection method has the disadvantages of low detection rate and long incubation time. The traditional serological method is still a common method in various laboratories because of its simplicity, rapidity and good sensitivity. Only an indirect sign of infection, diagnostic serological results are difficult to obtain when the body is immunocompromised
Due to the prevalence of Toxoplasma gondii infection in the population, the diagnostic value of serological testing for current infection is limited
Conventional PCR is easy to cause pollution during operation, and its high false positives make it subject to some limitations in clinical diagnosis. Therefore, an accurate, sensitive, fast, and pollution-free clinical testing method is urgently needed

Method used

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  • Fluorescent quantitative PCR (FQ-PCR) kit for rapidly detecting Toxoplasma gondii
  • Fluorescent quantitative PCR (FQ-PCR) kit for rapidly detecting Toxoplasma gondii

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: kit composition and preparation

[0036] (1) DNA extraction solution: Prepare lysis buffer, including the following components: 0.1M Tris-HCl (pH8.0), 0.1-0.15M NaCl, 0.1-0.5M EDTA (pH8.0) and 1%-4% SDS. Prepare a stock solution of proteinase K with a concentration of 20 mg / ml. When extracting tissue or cell genome, add proteinase K into the lysis buffer solution, the final concentration is 4ug / ul, which is the DNA extraction solution.

[0037] (2) Fluorescent quantitative PCR reaction solution: prepare reaction solution SYBR-Green I (10×) 0.5 μl, 10× buffer 2.5 μl, dNTP (2.5mmol / L) 2 μl, Taq enzyme (5U / μl) 0.2 μl, MgCl 2 (2.5mmol / L) 3.5μl, forward primer and reverse primer each 1μl (10μmol / L), sterile double distilled water 14.3μl.

[0038] (3) Standard positive template stock solution: the concentration is 10 10 Copy / μl standard positive template pMD-B1.

[0039] (4) Negative charge standard: sterile double distilled water.

Embodiment 2

[0040] Embodiment 2: the specificity test of kit

[0041] Use 1 μl each of 5 control positive samples, including Trichomonas, Giardia, Cryptosporidium parvum, Leishmania, and Eimeria tenena, which have been verified by DNA validity, as templates for fluorescent quantitative PCR reactions, and set a blank control at the same time group and positive control group.

[0042] The PCR amplification conditions were cycle conditions: pre-denaturation at 95°C for 60s; 15s at 95°C, 15s at 58°C, 45s at 72°C, and 40 cycles of amplification.

[0043] The results showed that only Toxoplasma had amplification curves, while Trichomonas, Giardia, Cryptosporidium parvum, Leishmania, and Eimeria tena had no amplification curves. Toxoplasma was repeated three times, and the melting temperature of the melting curve was stable, indicating that the specificity of the PCR amplification product of the target gene was better; the obtained PCR products were subjected to agarose gel electrophoresis, and...

Embodiment 3

[0044] The sensitivity test of embodiment 3 kits

[0045] Firstly, the counted Toxoplasma gondii tachyzoite DNA was diluted to detect its total DNA content, and the double ratio method was used to make 10×, 100×, 1000×, 2000×, 4000×, 8000× diluted Toxoplasma gondii DNA, each 1 μl was The template was subjected to fluorescence quantitative PCR reaction, and a blank control group was set at the same time.

[0046] The PCR amplification conditions were cycle conditions: pre-denaturation at 95°C for 60s; 15s at 95°C, 15s at 58°C, 45s at 72°C, and 40 cycles of amplification.

[0047] The sensitivity of the kit was determined by the experimental results, and the amplification curves of 6 dilutions showed that the fluorescent quantitative PCR kit could detect up to 0.5 Toxoplasma gondii.

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Abstract

The invention discloses a fluorescent quantitative PCR (FQ-PCR) kit for rapidly detecting Toxoplasma gondii, relating to gene detection technology of zoonosis pathogen, and being applicable to qualitative and quantitative detection of Toxoplasma gondii. The invention comprises DNA extracting solution, a standard positive template, a fluorescent quantitative PCR reaction solution, Toxoplasma gondii B1 gene specific primer and a negative quality control standard product. The invention is accurate in quantifying, rapid in detection speed, good in specificity, high in sensitivity, simple in use steps and high in repeatability and can replace traditional etiology detection method.

Description

technical field [0001] The invention belongs to the technical field of detecting pathogens of zoonotic infectious diseases, in particular to a fluorescent quantitative PCR kit for rapid detection of toxoplasma gondii, which is suitable for qualitative and quantitative detection of toxoplasma gondii. Background technique [0002] Toxoplasma gondii is a parasitic protozoan widely parasitic in the nucleated cells of humans and animals, with cats as its final hosts and humans and many vertebrates as intermediate hosts. The worm is distributed worldwide and can infect humans and many animals, causing zoonotic toxoplasmosis. Due to its great potential harm to human health and agricultural and animal husbandry production, it is very important to diagnose toxoplasmosis quickly and accurately. [0003] In terms of clinical detection, the traditional etiological detection method has the disadvantages of low detection rate and long incubation time. The traditional serological method i...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64C12R1/90
Inventor 刘全商立民
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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