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Primers, probes and method for detecting plasmodium

A Plasmodium and probe technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of time-consuming and labor-intensive blood smear inspection methods, complex detection condition settings, low specificity and sensitivity, etc. , to achieve the effect of fast detection speed, stable results and high sensitivity

Inactive Publication Date: 2014-07-09
河北国际旅行卫生保健中心
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, there are more or less defects in these methods: for example, the blood smear examination method is time-consuming and laborious, and when the protozoa density is less than 50 protozoa / μl blood, it is difficult to detect by microscopic examination, and it is easy to miss the diagnosis
However, the existing detection kits provide primers and probes, or have low specificity and sensitivity, or have false positive results, or the detection steps are cumbersome, the detection conditions are complicated, or the labor cost is high. Therefore, the design The method with simple detection steps, low cost, high sensitivity and specificity, and the primers and probes with high specificity and sensitivity, which are conducive to the setting of experimental conditions, will improve the stability, accuracy and quality of the method for detecting malaria parasites and their species. Practicality is of great significance and has become one of the issues that need to be improved

Method used

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  • Primers, probes and method for detecting plasmodium
  • Primers, probes and method for detecting plasmodium

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Design and synthesis of primers and probe sequences

[0029] Search for the sequence of the target gene SSUrRNA of the malaria parasite pathogen from Genebank, use relevant bioinformatics software for homology analysis and BLAST sequence analysis, screen out highly conserved regions, and use the primer design software Primer Premier 5 for the conserved sequences to carry out primers and probes After the design of primers and probes, use NCBI BLAST to test the specificity, and use Primer premier 5 to detect whether there is a hairpin structure or dimer formation. The results show that the designed probes meet the specificity requirements and will not form Hairpin structure or dimer. The designed primers and probe sequences are listed in Table 1.

[0030] Table 1 Primers and probes used to detect Plasmodium

[0031] name sequence SEQ ID No: PU-F2 TTGTTGCAGTTAAAACGCTCG 1 PU-R2 GCTTTGAACACTCTAATTTACTC 2 PV-PF2 GCAACGCTTCTTA...

Embodiment 2

[0032] Example 2 . Preparation of Four Plasmodium-specific Probe Microsphere Sets

[0033] 1. Probe synthesis

[0034] Various probe sequences were synthesized according to SEQ ID No: 3-6 in Table 1, and the 5' ends of all probes were modified with amination and connected with a C12 adjacent arm sequence.

[0035] 2. Coupling of probes and microspheres

[0036] Randomly select 4 different fluorescent microspheres, the numbers of the selected fluorescent microspheres in this embodiment are 45, 49, 52, 56, provided by BioRad, and the nucleic acid probes are coupled according to the following method:

[0037] 1. Put 4 types of microspheres numbered 45, 49, 56, and 52 stored at 2-10°C and 2 bottles of unopened EDC powder (10mg / bottle) stored at -20°C at room temperature for 30-60 minutes;

[0038] 2. The PV-PF2, PM-PF2, PO-PF2, PF-PF2 nucleic acid probes were respectively separated by ddH 2 O is dissolved at a concentration of 0.1 nmol / μl;

[0039] 3. Vortex the microsphe...

Embodiment 3

[0062] Example 3 : Detection of Plasmodium in 4 clinical samples

[0063] 1. Sample preparation

[0064] The genomic DNA of No. 1-4 clinical samples was extracted by conventional methods, and the concentration was measured for future use. The clinical samples could be serum or mosquitoes. In this embodiment, human serum was selected as the test sample.

[0065] 2. Multiplex PCR amplification

[0066] 1. Synthesize PCR amplification primers according to SEQ ID No: 1-2 in Table 1, wherein the 5' ends of the downstream primers all have biotin modification

[0067] 2. PCR amplification reaction:

[0068] (1) PCR reaction system: PCR amplification reaction system is 20 μl, including 10×PCR Buffer 2 μl, dNTP (2.5 mmol / L) 2 μl, goldTaq enzyme (5U / μl) 0.07 μl, PU-F2 (5 μM) 0.3 μl , PU-R2 (5μM) 1.5μl, genomic DNA 1μl, add ddH 2 O 11.6μl to a final volume of 20μl; at the same time, set up a negative control group, with 1μl ddH 2 O is a template instead of genomic DNA;

[0...

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Abstract

The invention discloses primers, probes and a method for detecting plasmodium, belonging to the technical fields of biological detection and biological chemistry. The adopted technical scheme is as follows: the invention provides a method for detecting plasmodium, universal primers SEQ ID No: 1-2 for PCR (Polymerase Chain Reaction) amplification of gene sequences of plasmodium in the method, and probes SEQ ID No: 3-6 for detecting plasmodium. The primers, probes and method have the advantages that (1) the universal primers for PCR amplification of the gene sequences of plasmodium and the specific nucleic acid probes are high in sensitivity and good in accuracy; (2) due to the adoption of an asymmetric PCR method and liquid chip combined technology, the yield of a single strand of a biological marker is increased so that the hybridization and combination efficiencies of PCR products and microspheres coupled with the specific nucleic acid probes are increased; the method has the advantages of high-flux test, high specificity and sensitivity, stable result, good repeatability, simplicity in operation, high detection speed and capability of rapidly detecting plasmodium.

Description

technical field [0001] The invention belongs to the technical fields of biological detection and biochemistry, and in particular relates to a primer, a specific nucleic acid probe and a detection method capable of rapidly and accurately detecting / identifying malaria parasites. Background technique [0002] Malaria is a parasitic disease that seriously endangers human health. It spreads to more than 90 countries and regions around the world, threatening 41% of the world's population. Together with AIDS and tuberculosis, it is listed by the World Health Organization as one of the three most serious infectious diseases threatening human health. There are many kinds of Plasmodium, and there are 4 kinds of Plasmodium that parasitize humans, and the clinical manifestations and symptoms caused by them are similar: generally intermittent chills, fever, sweating, sometimes causing splenomegaly and anemia; severe patients can cause cerebral palsy. , liver, kidney and other organ da...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/686C12Q2537/143C12Q2563/149Y02A50/30
Inventor 闫冀焕李云史玲莉滑娜沈军吴志茹兰景李薇付晓昀
Owner 河北国际旅行卫生保健中心
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