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Method for quantitatively detecting hantaan virus load through one-step fluorescence probe real-time quantitative reverse transcription polymerase chain reaction

A technology of reverse transcription polymerase and fluorescent probe, applied in the direction of microbial measurement/testing, microbial-based methods, biochemical equipment and methods, etc. Beach virus and other problems, to achieve the effect of high sensitivity, reduced operation steps, high sensitivity

Inactive Publication Date: 2012-08-15
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

One-step real-time fluorescent probe quantitative reverse transcription polymerase chain assay for the load of other Hantavirus types including Dobrava Vires, Puumala Vires and Sin Nombre Vires There are research reports on the reaction detection method, but there is no research on the relationship between Hantaan virus load and disease at home and abroad, and there is no one-step fluorescent probe real-time quantitative reverse transcription polymerase chain reaction method for quantitative detection of Hantaan virus load research report

Method used

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Examples

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Embodiment 1

[0033] Example 1: Quantitative detection of Hantaan virus load in plasma of patients with hemorrhagic fever with renal syndrome:

[0034] Use the plasmid containing the 76-118S segment of the Hantaan virus standard strain to transcribe in vitro to obtain the S segment RNA of the 76-118 strain, quantify it accurately with an ultraviolet spectrophotometer, and calculate the molar concentration per unit volume based on its molecular weight as a standard; adjust the standard Concentration is 10 10 copies / ml, and 10 gradients were diluted 1:10 times as a standard curve.

[0035] After design and screening of primers and probes, the reverse transcription primers are random hexamers, in which:

[0036] The upstream primer is 5'-TACAGAGGGAAATCAATGCC-3';

[0037] The downstream primer is 5'-TGTTCAACTCATCTGGATCCTT-3';

[0038] The probe was 5'-(FAM)ATCCCTCACCTTCTGCCTGGCTATC(TAMRA)-3'.

[0039] Quantitative in vitro transcribed RNA was used as a standard, RNase-free water was used as...

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Abstract

The invention discloses a method for quantitatively detecting hantaan virus load through one-step fluorescence probe real-time quantitative reverse transcription polymerase chain reaction. According to the method, the overall-length fragment of in vitro transcription S of the hantaan virus is utilized as a quantitative detection standard substance, a one-step fluorescence probe real-time quantitative reverse transcription polymerase chain reaction method is established by the design of detecting a virus gene sequence primer and a prober and the selection of the method so as to quantitatively detect the hantaan virus load of the blood plasma or other body fluid and tissues of a hemorrhagic fever with renal syndrome patient. Compared with a conventional PCR (Polymerase Chain Reaction) detection method, the method has the advantages that the specimen detection and operation steps are few, the sensitivity is high, the linear relation is good, and the experiment shows that the detection method has good specificity and repeatability and provides a powerful tool for the scientific research and preventive treatment of hemorrhagic fever with renal syndrome.

Description

technical field [0001] The invention belongs to the technical field of molecular biology analysis, and relates to a method for quantitatively detecting viruses, in particular to a method for quantitatively detecting Hantaan virus load in real time with a one-step fluorescent probe by reverse transcription polymerase chain reaction. Background technique [0002] Hemorrhagic fever with renal syndrome (HFRS) is an acute infectious disease characterized by fever, hemorrhage, hypotensive shock and renal failure caused by certain serotypes of Hantavirus. . HFRS is widely prevalent in the world, and the epidemic in China is the most serious, accounting for about 90% of global cases, and the mortality rate of the disease is about 1% to 15%. The main popular types in my country are Hantaan Virus and Seoul Virus. The basic pathological changes of hemorrhagic fever with renal syndrome are extensive damage and increased permeability of small blood vessels and capillaries throughout th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 易静庄然徐竹蔚张赟張春梅马樱刘蓓张宇丝陈丽华杨琨宋朝君李琦方亮周幸春刘志佳金伯泉
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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