Fluorescent quantitative PCR (FQ-PCR) kit for rapidly detecting Leishmania donovani

A Leishmania donovani, fluorescence quantitative technology, applied in fluorescence/phosphorescence, microorganism-based methods, and microbial determination/inspection, etc., can solve the problems of long incubation time, low detection rate, PCR contamination, etc. Simple steps to use, fast detection speed, good repeatability

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

AI Technical Summary

Problems solved by technology

[0005] In terms of clinical detection, the traditional etiological detection method has the disadvantages of low detection rate and long culture time. The traditional serological method is still a common method in various laboratories because of its simplicity, rapidity and good sensitivity. However, serological Only an indirect sign of infection, diagnostic serological results are difficult to obtain when the body is immunocompromised
Moreover, due to the prevalence of Leishmania donovani infection in the population, the diagnostic value of serological testing for current infections 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 Leishmania donovani
  • Fluorescent quantitative PCR (FQ-PCR) kit for rapidly detecting Leishmania donovani

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: kit composition and preparation

[0037] (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.

[0038] (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.

[0039] (3) Standard positive template stock solution: the concentration is 10 10 Copy / μl standard positive template pMD-kDNA.

[0040] (4) Negative charge standard: sterile double distilled water.

Embodiment 2

[0041] Embodiment 2: the specificity test of kit

[0042] Use 1 μl each of five control positive samples, including Trichomonas, Giardia, Cryptosporidium parvum, Toxoplasma gondii, and Eimeria tenena, which have been verified by DNA validity, as templates for fluorescent quantitative PCR reactions. positive control group.

[0043] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 60s; 15s at 95°C, 15s at 56°C, 45s at 72°C, and 40 cycles of amplification.

[0044] The results showed that only Leishmania had amplification curves, while Trichomonas, Giardia, Cryptosporidium parvum, Toxoplasma gondii and Eimeria tenella had no amplification curves. After three repetitions of Leishmania, 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 all of them were similar in size to the...

Embodiment 3

[0045] The sensitivity test of embodiment 3 kits

[0046] Firstly, dilute the counted Leishmania promastigotes DNA, detect its total DNA content, and make 10×, 100×, 1000×, 2000×, 4000×, 8000× diluted Leishmania DNA , 1 μl of each was used as a template for fluorescent quantitative PCR reaction, and a blank control group was set at the same time.

[0047] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 60s; 15s at 95°C, 15s at 56°C, 45s at 72°C, and 40 cycles of amplification.

[0048] 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 Leishmania.

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Abstract

The invention discloses a fluorescent quantitative PCR (FQ-PCR) kit for rapidly detecting Leishmania donovani, relating to gene detection technology of zoonosis pathogen, and being applicable to qualitative and quantitative detection of Leishmania donovani. The invention comprises a standard positive template, a fluorescent quantitative PCR reaction solution, Leishmania donovani kDNA gene specific primer and 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 Leishmania donovani, which is suitable for qualitative and quantitative detection of Leishmania donovani. Background technique [0002] Kala azar (Visceral Leishmaniasis, VL) is caused by Leishmania donovani and is a zoonotic infectious disease with sandflies as the medium. Clinically, it is characterized by long-term irregular fever, hepatosplenomegaly, anemia and peripheral blood leukopenia. as the main feature. In recent years, the co-infection of VL with tuberculosis and AIDS has been increasing year by year. [0003] Sandfly chinensis is the main vector of VL in my country, with a wide distribution. Generally, it begins to appear in early May, and only one generation is reproduced a year. The peak is mostly seen in June, and only a few areas will have a small peak in July ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64C12R1/90
CPCY02A50/30
Inventor 刘全商立民
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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