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Chimeric primer multiple PCR molecular detection kit for malaria species-genera detection and detection method thereof

A technology of chimeric primers and molecular detection, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, resistance to vector-borne diseases, etc. Specific identification and other issues, to achieve the effect of improving detection sensitivity and specificity, clean background, and less non-specific amplification

Active Publication Date: 2016-06-22
SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, only one target gene can be amplified in one reaction, which is not suitable for the diagnosis of malaria parasites with multiple species; nested PCR can be used to identify multiple species, but two rounds of PCR reactions are required, each round of reaction Requires multiple reactions to complete, time consuming and laborious
Fluorescent quantitative PCR has high sensitivity, but the cost of equipment and reagents is also high, which is not conducive to grass-roots use
LAMP does not require special equipment, and the results can be observed with the naked eye, but it cannot be used to identify insect species

Method used

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  • Chimeric primer multiple PCR molecular detection kit for malaria species-genera detection and detection method thereof
  • Chimeric primer multiple PCR molecular detection kit for malaria species-genera detection and detection method thereof
  • Chimeric primer multiple PCR molecular detection kit for malaria species-genera detection and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, chimeric primer multiple PCR detection system and method for the same detection of malaria species

[0072] 1.1 Design of chimeric primers

[0073] The ribosomal 18SrRNA and mitochondrial genes of Plasmodium were selected as target sequences. A total of five pairs of Plasmodium and species-specific primers were designed using GenBank public database resources (http: / / blast.ncbi.nlm.nih.gov / Blast.) and primer design software PrimerPrimer5.0 (Table 1). Among them, the amplified product of Plasmodium-specific primers is a conserved segment of mitochondrial genes, and the specific primers of four human Plasmodium species amplify ribosomal 18SrRNA-specific segments respectively. The gene-specific primers in Table 1 were respectively connected to a common non-specific nucleic acid fragment (universal primer, 5'-CGAGTCCTGCGGTCTCAAATT-3'). The 3' end of each primer is a sequence-specific recognition site, and the 5' end is a 21bp unrelated sequence to form a chi...

Embodiment 2

[0084] Example 2, Sensitivity and specificity analysis of chimeric primer multiplex PCR detection system for the same detection of malaria species

[0085] 2.1 Counting of protozoa

[0086] Whole blood samples were coated with thick and thin blood films, dried, fixed in methanol, and then stained with Giemsa. Plasmodium was checked and counted by experienced microscope examiners. The method of counting malaria parasites is to count more than 200 white blood cells, and count 1000 white blood cells when the density is very low. The density of malaria parasites was calculated by the following formula: number of malaria parasites ÷ number of white blood cells × number of white blood cells per μL of blood = number of malaria parasites / μL of blood (if white blood cell counts cannot be counted, calculate as 8000 white blood cells / μL of blood).

[0087] 2.2 Sensitivity of multiplex PCR detection of blood samples with different protozoa densities

[0088] Select 3 copies of each of t...

Embodiment 3

[0096] Embodiment 3, multiplex PCR detection malaria patient and normal person's blood sample

[0097] The detection effect of single-blind method on 20 blood samples of malaria patients (8 samples of P. Figure 5 shown. Except for 2 samples of malaria malaria with slight miscellaneous bands, the bands of other malaria samples were clear and the background was clean. There was no obvious cross-reaction in normal blood samples. In the multiplex PCR detection system, different "band types" formed by the combination of genus-specific bands and species-specific bands make the results clear at a glance and easy to judge.

[0098] The chimeric primer multiple PCR molecular detection kit and its detection method of the same detection of malaria species in the invention are adopted, and its technical effect is:

[0099] 1) Four species of human Plasmodium can be determined in one reaction. For a single Plasmodium infection sample, two specific gene fragments of the genus and speci...

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Abstract

The invention relates to a chimeric primer multiple PCR molecular detection kit for malaria species-genera detection and a detection method thereof. The kit comprises a mixed primer combination, a 2*multiple PCR premix, a multiple PCR positive reference and a 100bp molecular weight Marker, wherein the mixed primer combination comprises a specific chimeric primers with sequences of SEQ No.1-SEQ ID No.10 and a universal primer with sequence of SEQ ID No.11; and the detection method comprises a multiple PCR amplification reaction, wherein the circulating temperature refers to three circulating annealing temperatures of 58 DEG C, 62 DEG C and 68 DEG C increasing step by step. By adopting the chimeric primer multiple PCR molecular detection kit for malaria species-genera detection and the detection method thereof, through the combination of the primer sequences shown by SEQ ID No.1-11, the gene segments of the specific species and genera in a malaria sample can be amplified at the same time by once PCR reaction, and four kinds of plasmodium can be identified and diagnosed according to the size and combination of the amplified segments. The time and human and material resources are saved, and the detection efficiency is improved.

Description

technical field [0001] The invention relates to the field of medical reagents, in particular to a genetic diagnosis kit, in particular to a chimeric primer multiplex PCR molecular detection kit and a detection method for the same detection of malaria species. Background technique [0002] Malaria is a serious parasitic disease caused by Plasmodium infection. There are four main species of Plasmodium that infect humans: Plasmodium falciparum (Pf), Plasmodium vivax (Pv), Plasmodium malariae (Pm) and Plasmodium ovale (P. ovale, Po). According to the latest WHO data, globally, approximately 3.3 billion people in 97 countries and regions are at risk of malaria infection and other diseases it causes. There were 198 million malaria cases worldwide in 2013, resulting in 584,000 deaths. Of these, 90% of the deaths occurred in Africa, and children under the age of 5 accounted for 78% of all deaths. In my country, the current malaria epidemic situation has undergone significant cha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q1/6893C12Q2600/16C12Q2537/143Y02A50/30
Inventor 江莉蔡黎张耀光王真瑜朱民马晓疆吴寰宇
Owner SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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