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Novel Kit for fluorescence quantitative PCR detection of influenza A H1N1 viruses and detecting method therefor

A detection method and fluorescence quantitative technology are applied in the detection field of a new type A H1N1 virus by fluorescence quantitative PCR, and can solve the problems of high detection cost, detection specificity, limited sensitivity and low sensitivity detection.

Inactive Publication Date: 2010-03-10
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the relevant sequences published by NCBI were limited at that time, and the primers and probes published by WHO had limited specificity and sensitivity for the detection of this outbreak of H1N1 influenza virus.
[0005] The present invention is aimed at the relatively low sensitivity detection and high detection cost of currently designed and published primers and probes.

Method used

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  • Novel Kit for fluorescence quantitative PCR detection of influenza A H1N1 viruses and detecting method therefor
  • Novel Kit for fluorescence quantitative PCR detection of influenza A H1N1 viruses and detecting method therefor
  • Novel Kit for fluorescence quantitative PCR detection of influenza A H1N1 viruses and detecting method therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Detection of Primer Sensitivity

[0037] 1) Select the full length of the known sample HA gene as a standard, and increase its viral RNA concentration from 10 -1 sequentially diluted to 10 -10 ;

[0038] 2) Negative control: nuclease-free water;

[0039] 3) The reaction system was prepared according to Table 3.

[0040] Test results such as figure 2 , 3 shown.

[0041] figure 2 The curves 1-6 in the middle are respectively the concentration dilution of the standard substance is 10 -4 -10 -10 Amplified curve with primer WHO-SWH1. image 3 The curves 1-6 in the middle are the concentration dilution of the standard substance in order of 10 -4 -10 -10 Curve amplified with primer YHANEW. we start from figure 2 and image 3 It can be seen from the figure that the curve of the amplification result of the YHANEW primer is earlier than the curve of the amplification result of the WHO-SWH1 primer.

[0042] Table 4SWH1, YHANEW primer sensitivity detect...

Embodiment 2

[0045] Example 2: Primer non-characteristic detection

[0046] 1) Non-specific detection of common seasonal influenza H1N1, swine influenza H1N1, avian influenza H5N1, H9N2;

[0047] 2) Positive control: viral HA fragment RNA;

[0048] 3) Negative control: nuclease-free water;

[0049] 4) The reaction system was prepared according to Table 2.

[0050] See the test results Figure 4 and Table 5.

[0051] Figure 4 The results showed that only the positive control HA numbered 1 showed an amplification curve. The curves of the remaining samples were all below the baseline, and there was no amplification reaction.

[0052] Table 5 Human, poultry and swine influenza virus detection results

[0053]

[0054] The data in Table 5 shows that the primers have a CT value of "-" for gene amplification of common seasonal influenza H1N1, swine influenza H1N1, avian influenza H5N1 and H9N2. It shows that the primer has no amplification reaction to its viral RNA.

Embodiment 3

[0055] Embodiment 3: detection of patient's throat swab

[0056] 1) Detect the throat swabs collected from suspected cases to screen for the new type A H1N1 influenza virus;

[0057] 2) Positive control: viral HA fragment RNA;

[0058] 3) Negative control: nuclease-free water;

[0059] 4) The reaction system was prepared according to Table 3.

[0060] See the test results Figure 5 and Table 6.

[0061] Figure 5 Only one amplification curve appeared in the positive control, and the rest of the samples had no amplification. The reaction was negative.

[0062] Table 6 Throat swab test results

[0063]

[0064] The results of the data in Table 6 show that the results of the patients' throat swabs were all negative.

[0065] Result analysis:

[0066] After the amplification reaction completed 40 cycles, the primer sensitivity detected that the viral RNA concentration dilution was 10 -8 ; There is no cross-reaction with common seasonal influenza H1N1, swine influenza ...

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Abstract

The invention discloses a novel kit for fluorescence quantitative PCR detection of influenza A H1N1 viruses, containing a primer, a probe, Taqman 2*universal PRC master mix, 40*multi scribe<TM> and RNase inhibitor mix and nuclease-free water. The primer and the probe have the advantages of good detecting specificity and high sensitivity so that the kit is particularly suitable for the influenza AH1N1 which breaks out at present and has no cross reaction with the Avian flu, swine flu and common seasonal flu and has lower cost.

Description

technical field [0001] The invention provides a fluorescent quantitative PCR detection method, detection primers and specific probes for the popular influenza A (H1N1) virus. Background technique [0002] Since April 2009, outbreaks of human infection with influenza A (H1N1), a new influenza A virus, have occurred in many countries, including Mexico, the United States, and Canada. As the number of people infected with Influenza A (H1N1) continues to increase, the epidemic is becoming more and more serious. On June 11, WHO raised the level of influenza pandemic alert to level 6. The world is at the beginning of the 2009 influenza pandemic. Influenza A (H1N1) virus is a single-stranded RNA virus of the Orthomyxoviridae Influenza A virus genus. Influenza A viruses can be divided into multiple subtypes according to the different antigenic properties of their external glycoproteins hemagglutinin (HA) and neuraminidase (NA). HA and NA are highly variable but extremely conserved ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12R1/93
Inventor 姚李四胡孔新燕清丽孙肖红杨鹏飞王静
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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