Plasmodium falci parum nano magnetic separation real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit and nucleotide sequence

Abstract

The invention relates to a plasmodium falci parum nano magnetic separation real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit and a nucleotide sequence. The invention designs plasmodium falci parum-specific primers and probes, and optimizes the real-time fluorescence PCR detection system. A pair of primers for plasmodium falci parum are combined with a common PCR technique and a molecular cloning technique to construct a plasmodium falci parum plasmid standard substance, establish a standard curve about the relationship between the target gene copy number and fluoroscopic examination signals and implement real-time fluorescence quantitative detection of plasmodium falci parum. By organically combining the technique of separating plasmodium nucleic acid by nano magnetic particles with the real-time fluorescence quantitative PCR technique, the kit is convenient to operate and is cheap, quick and efficient in the aspect of specimen nucleic acid extraction, has great advantages in the aspects of filter paper dried blood spot nucleic acid extraction and trace whole blood nucleic acid extraction, and achieves the goals of widening the detection range and maximally lowering the omission factor. The detection limit of the kit is 25 copies / mu L.

Description

technical field [0001] The invention belongs to the field of inspection and quarantine, but is not limited to the field, relates to the detection of Plasmodium falciparum in clinical samples, and is a real-time fluorescent quantitative PCR detection kit and nucleotide sequence for Plasmodium falciparum magnetic separation in nanometers. Background technique [0002] Malaria is one of the most serious infectious diseases to human health. 86% of malaria cases occur in Africa, and 80% of malaria cases outside Africa are concentrated in India, Sudan, Myanmar, Bangladesh, Indonesia, Papua New Guinea and Pakistan other countries. Plasmodium falciparum is the main species in Africa, and Plasmodium vivax is the most widely distributed in tropical, subtropical, and temperate countries and regions, especially in Central America and Southeast Asia. [0003] Since 2000, the malaria epidemic in my country has rebounded, especially in Yunnan and Hainan provinces. Many provinces are threa...

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Problems solved by technology

The boiling method is simple and economical, but the nucleic acid yield is low and the purity is low, which is not conducive to the long-term preservation of the nucleic acid, and the nucleic acid extract contains hemoglobin and other substances that inhibit the nucleic acid amplification reaction, which affects the detection rate of malaria parasites; commercial kits The extraction rate of nucleic acid is high, and there are fewer factors affected during nucleic acid amplification, but the kit is expensive, and it is not suitable for large-scale use on site or in remote and poor areas

[0011] Other Plasmodium nucleic acid extraction methods include sodium iodide method, phenol chloroform extraction method, etc. These methods are time-consuming, use toxic reagents, are harmful to human body, pollute the environment, and are not suitable for routine detection

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