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General purpose real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) non-diagnostic method for Hantaan virus detection

A RT-PCR, Hantavirus technology, applied in fluorescence/phosphorescence, biochemical equipment and methods, microbial determination/inspection, etc., can solve single problems, achieve high application value, and reduce the chance of amplification product contamination , high sensitivity and specificity

Inactive Publication Date: 2013-02-06
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no universal detection method for hantaviruses, basically based on each hantavirus genotype, a single amplified corresponding type

Method used

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  • General purpose real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) non-diagnostic method for Hantaan virus detection
  • General purpose real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) non-diagnostic method for Hantaan virus detection
  • General purpose real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) non-diagnostic method for Hantaan virus detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Detection of Primer Sensitivity

[0056] Select several representative hantavirus types such as Hantaan type (HTNV), Seoul type (SEOV), Puumala type (PUUV), Dobrava-Belgrade type (DOBV) and Sin Noble type (SNV) ) of the L full-length gene in the highly conserved region fragment as a standard, its concentration from 10 -3 sequentially diluted to 10 -10 ;

[0057] 1) Negative control: Hantavirus-negative mouse RNA;

[0058] 2) NTC: nuclease-free water

[0059] 3) The reaction system was prepared according to Table 3.

[0060] 4) The PCR reaction system is:

[0061]

[0062] The test results are illustrated by taking the Hantaan virus (HTNV) as an example, as follows: Figure 4 shown.

[0063] Figure 4 The curves 1 to 8 in the middle represent the dilution of the standard product with nuclease-free water concentration of 10 -3 ~10 -8 Amplified curve. In summary, it can be concluded from the figure that the minimum detection limit of the primer and p...

Embodiment 2

[0068] Embodiment 2: the making of universal hantavirus real-time fluorescent PCR standard curve

[0069] 1) Select Puumala-type hantavirus as a standard substance, and its concentration is sequentially diluted to 10 -10 , select 10 -3 ~10 -10 as a template;

[0070] 2) Negative control: Hantavirus negative mouse lung RNA;

[0071] 3) The PCR reaction system is

[0072]

[0073] The result is as Figure 5 As shown, the standard curve of hantavirus universal real-time fluorescent PCR is: y=37.860-3.220x, R 2 =0.996.

Embodiment 3

[0074] Embodiment 3: non-specific detection

[0075] 1) Select Dobrava-Belgrade type Hantavirus as positive control;

[0076] 2) Negative control: nuclease-free water;

[0077] 3) Template: select hantavirus-negative mice for non-specific testing, and the mouse species include Apodemus striatus, Apodemus spp., Rattus norvegicus, Rattus norvegicus, etc.

[0078] 4) Prepare reaction system, same as above.

[0079] The result is as Figure 6 , the established general-purpose real-time fluorescent PCR method for hantavirus has no specific amplification for the host RNA of hantavirus.

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Abstract

The invention discloses a general purpose real-time fluorescent RT-PCR (reverse transcription-polymerase chain reaction) non-diagnostic method for Hantaan virus detection. The detection method comprises the following steps: selecting a Hantaan virus L gene full-length sequence, performing homologous alignment, and designing primers in a conserved region; designing a specific probe according to a nucleotide sequence in a PCR amplification region, and detecting the specificity of the probe; and performing real-time quantitative analysis on an object to be detected. The specific fluorescent probe is used to perform a completely closed pipe type operation, so that the possibility of polluting the amplification product is greatly reduced, and experiment steps, experiment time and the consumption of reagents are reduced. At present, no general purpose Hantaan virus detection method exists, and the existing method is basically used for singly amplifying one corresponding type based on one Hantaan genotype. According to the invention, the pair of primers and the probe are used to detect Hantaan viruses of different genotypes, the real-time PCR has very high sensitivity and specificity, and the detection of a Hantaan virus gene is sensitive and accurate. In practice, the method has a high application value especially in case of an epidemic outbreak.

Description

technical field [0001] The invention provides a non-diagnostic method for detecting hantavirus by using fluorescent quantitative RT-PCR. Background technique [0002] Hantavirus belongs to the genus Hantavirus (Hantavirus) of the family Bunyaviridae (Bunyaviridae). It consists of three gene segments, encoding RNA-dependent RNA polymerase, envelope glycoproteins Gn, Gc, and nucleoprotein, respectively. There are many genotypes of Hantavirus, more than 40 kinds have been discovered so far. Hantaviruses cause hemorrhagic fever with renal syndrome and hemorrhagic fever with pulmonary syndrome in humans. [0003] With global economic integration and trade liberalization, foreign medical vectors can quickly spread infectious diseases from one country or region to the world through advanced means of transportation and international trade, resulting in international spread. The widespread spread and prevalence of infectious diseases in the world has become a political issue that ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 杨鹏飞胡孔新燕清丽姚李四张丽萍
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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