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Folding primer multiple PCR malaria molecule diagnostic kit and detection method thereof

A folding primer and molecular diagnosis technology, applied in the field of medical testing, can solve the problems of complicated operation, easy contamination and mistakes, long test time, etc., and achieve the effect of enhancing sensitivity and specificity, improving efficiency, and avoiding non-specific amplification.

Inactive Publication Date: 2015-09-16
贵州省疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Purpose of the present invention: in order to overcome existing nested PCR and detect 4 kinds of malaria parasite diagnostic experiment simultaneously, need multi-tube two-step amplification, test time is long and reagent, operation is complicated, need middle opening tube to add sample again, easy to pollute and Mistakes; and the rapid PCR of single-tube one-step reaction can only detect and identify the defects of two kinds of Plasmodium PF and PV; provide a single-tube one-step amplification that can simultaneously detect and identify single and mixed infections of 4 kinds of Plasmodium, Sensitive, accurate, simple and fast folding primer multiplex PCR malaria molecular diagnostic kit

Method used

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  • Folding primer multiple PCR malaria molecule diagnostic kit and detection method thereof
  • Folding primer multiple PCR malaria molecule diagnostic kit and detection method thereof
  • Folding primer multiple PCR malaria molecule diagnostic kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Embodiment 1, application folding primer multiplex PCR (FP-PCR) malaria molecular diagnostic kit detects and identifies 4 kinds of human Plasmodium-sensitivity detection, the results are shown in Figure 1-Figure 4 .

[0061] Preparation of blood samples on hemofilter paper simulated by Plasmodium density gradient infection: Plasmodium vivax (PV), Plasmodium falciparum (PF), Plasmodium ovale (PO) (including variants) and Plasmodium malaria ( PM) Infected patients (protozoa density 10000 worms / μl blood or more) contain 1 part of whole blood containing worms, and use EDTA anticoagulated whole blood of healthy people to serially dilute 10 times to contain 10000, 1000, 100, 10 and 1 worm per 1 μl The gradient of worms contains whole blood of worms. Use a pipette to quantitatively drip the Plasmodium density gradient blood filter paper blood sample containing worms (4μl / each 6mm diameter filter paper disc), and after natural air drying, divide into sealed tubes and store at...

Embodiment 2

[0078] Embodiment 2, application folding primer multiplex PCR (FP-PCR) malaria molecular diagnostic kit detects and identifies and simulates 4 kinds of human Plasmodium mixed infections, the results are shown in Figure 5 .

[0079] Preparation of blood samples on blood filter paper for simulating mixed infection of 4 kinds of Plasmodium: the possible density of different species of plasmodium mixed infection may vary greatly. Considering the importance of detecting low-density PF among them, use the density gradient filter paper of 4 kinds of parasites prepared in Example 1 The blood sample template simulates the mixed infection of 3 groups (respectively 2, 3 and 4 kinds of protozoa), and each group has 4 combinations, and a total of 12 mixed templates with different combinations of insect species and density are prepared. The highest and lowest values ​​of each combination Densities vary by a factor of 100. Half of the combinations use PF as the lowest density, and the othe...

Embodiment 3

[0086] Embodiment 3, specificity determination

[0087] According to the operation method of Example 1, 10 healthy human blood samples were tested repeatedly 7 times, and there was no amplified band except dimer below moderate. Therefore, it will not affect the detection results of malaria infection.

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Abstract

The invention discloses a folding primer multiple PCR malaria molecule diagnostic kit and a detection method thereof. The kit is composed of a magnesium-containing template buffer and a magnesium-free single combination reagent, and comprises a complete set of reagents for DNA preparation and a folding primer PCR reaction. The folding primer takes plasmodium Cyt b gene as a target gene, designing is carried out on plasmodium and specific site, sensitivity and singularity of four plasmodium detected by folding primer PCR can be obviously increased, and reagent and test operation can be simultaneously simplified. The kit of the present invention is employed for detection, a traditional reagent complex operation of nested-set / multiple PCR is simplified to a single reagent and a single tube for one step amplification reaction, four plasmodium individual or mixing infection can be detected and identified simultaneously, minimum of detected monocontamination can reach or approach 1 plasmodium / muml blood; and four protozoon can be simultaneously detected and accurately identified when the mixing infection is high, and low density difference is 100 times. The diagnostic kit and the detection method is a reliable means for definite diagnosis and discriminating of malaria in hospitals and laboratory in province and city.

Description

technical field [0001] The invention relates to a folding primer multiple PCR malaria molecular diagnosis kit and a detection method thereof, which belong to the field of medical examination. Background technique [0002] There are four types of Plasmodium that infect humans: Plasmodium falciparum (PF), Plasmodium vivax (PV), Plasmodium malariae (PM) and Plasmodium ovale (PO), which in turn cause Plasmodium falciparum, Plasmodium vivax, malaria and malaria ovale. Malaria, AIDS, and tuberculosis are listed as three major global public health problems. The first two types of malaria were prevalent in my country. Since 2010, the incidence of malaria in the country has dropped to less than 1 / 100,000 for three consecutive years. my country has entered the stage of malaria elimination. Four species of Plasmodium (including different strains, mixed infection and low-density parasites) have continuously entered my country. In 2012, overseas imports accounted for 91.5% of the report...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
CPCY02A50/30
Inventor 黄天谊黄雨婷张念恒佘丹娅
Owner 贵州省疾病预防控制中心
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