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Quickly-nested PCR (polymerase chain reaction) malaria molecular diagnosis reagent kit and detection method

A technology of molecular detection and reagent kit, which is applied in the field of medical testing, can solve the problems of complex reagent operation, easy pollution, and long test time, and achieve the effects of simplifying reagents and operating steps, improving sensitivity, and reducing costs

Inactive Publication Date: 2014-07-09
GUANGXI ZHUANG AUTONOMOUS REGION CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention: to overcome the traditional nested PCR malaria diagnostic test that requires multiple tubes and two-step amplification, the test time is long, and the reagent operation is complicated, the tube needs to be opened in the middle and the sample is easily polluted, and non-specific amplification such as dimer is seriously affected Defects such as result judgment; standardize reagents and operations, uniformly prepare combined reagents, and complete most of the reagent operations in the kit prefabrication stage to ensure rapid test, repeatability and reduce detection costs, providing a rapid nested PCR malaria Molecular diagnostic kit and detection method thereof

Method used

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  • Quickly-nested PCR (polymerase chain reaction) malaria molecular diagnosis reagent kit and detection method
  • Quickly-nested PCR (polymerase chain reaction) malaria molecular diagnosis reagent kit and detection method
  • Quickly-nested PCR (polymerase chain reaction) malaria molecular diagnosis reagent kit and detection method

Examples

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Embodiment 1

[0070] Example 1: Detection of Plasmodium falciparum and Plasmodium vivax using a rapid nested PCR malaria molecular diagnostic kit——sensitivity detection, see the results figure 2 .

[0071] Preparation of blood samples from blood filter papers simulated by density gradient infection of Plasmodium: collect one part of whole blood containing parasites from patients diagnosed with simple Plasmodium falciparum and simple Plasmodium vivax infection (above the density of protozoa 10000 worms / μl of blood) from veins, and use healthy Human anticoagulated whole blood was diluted with 10-fold serial dilution method to obtain 10000 / μl, 1000 / μl, 100 / μl, 10 / μl, 1 / μl gradient worm-containing blood, and use A pipette was used to quantitatively prepare a blood sample of Plasmodium-containing blood filter paper with a density gradient of plasmodia (5 μl / per piece of 6mm diameter filter paper disc), and after natural air drying, it was divided into sealed tubes and stored at 4°C for later us...

Embodiment 2

[0086] Example 2: Detection of Plasmodium falciparum and Plasmodium vivax by using the Rapid Nested PCR Malaria Molecular Diagnosis Kit - Detecting blood samples of malaria febrile patients in malaria surveillance, see the results image 3 .

[0087] Blood sample collection: Use the filter paper method to collect blood samples from clinically diagnosed malaria or suspected malaria patients. Routinely disinfected earlobe or fingertip blood pricking can be used. The center of the filter paper is directly in contact with the blood drop, so that the blood penetrates the filter paper to a diameter of 2-4cm. It is used after natural air drying. Put the white paper in a film bag at intervals and mail it at room temperature or store it at 4°C; or use a loose-leaf paper puncher to punch out small discs with a diameter of 6mm (containing about 4-5μl whole blood), and put them in a 0.5-1.5ml conical tube 4 Store at ℃.

[0088] Preparation of filter paper blood sample DNA template: use a...

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Abstract

The invention discloses a quickly-nested PCR (polymerase chain reaction) malaria molecular diagnosis reagent kit and a detection method. The quickly-nested PCR malaria molecular diagnosis reagent kit comprises novel primers with a self-closing function, cytochrome oxidase genes (cox1) in a plasmodium mitochondrial genome serve as target genes, and four bidirectional semi-nested primers are designed in loxp sites of plasmodium groups and species to form a pair of group-specific primers and two species-specific primers. A traditional nested / multiplex PCR complicated in reagents and burdensome in operation is simplified to single-reagent and single-tube one-step reaction, separate infection or mixed infection of malignant malaria and plasmodium vivax and separate infection of quartan malaria and ovale malaria can be detected and authenticated simultaneously, sensitivity and specificity of the detection method are superior to a microscopic examination method, and the level of detection of one worm / per microliter blood can be achieved. The detection method serves as a reliable means for diagnosis and differential diagnosis of malaria of hospitals at all levels, and the quickly-nested PCR malaria molecular diagnosis reagent kit is a simple, economic, sensitive and accurate malaria diagnosis tool for control and monitoring of malaria, malaria medicine tests, malaria vaccine research and blood donor screening.

Description

technical field [0001] The invention belongs to the field of medical testing, and relates to a rapid nested PCR malaria molecular diagnosis kit and a detection method thereof. Background technique [0002] Malaria is one of the three most serious infectious diseases (AIDS, tuberculosis and malaria) in the world. The World Health Organization (WHO) has listed malaria control as the action goal of this century. There are four kinds of Plasmodium that infect humans: Plasmodium falciparum, P.vivax, P.malariae and P.ovale. Due to the biological and immunological characteristics of the four Plasmodium species, their pathogenicity, therapeutic effect of antimalarial drugs, susceptibility to the vector Anopheles mosquitoes and their distribution in the regions of global prevalence vary. Every year, hundreds of millions of malaria patients are infected worldwide, and more than one million of them die from falciparum malaria. Timely and accurate diagnosis and differential diagnosis ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 黄天谊郭传坤黎学铭
Owner GUANGXI ZHUANG AUTONOMOUS REGION CENT FOR DISEASE CONTROL & PREVENTION
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