Fluorescent quantitative pcr detection kit and non-diagnostic detection method for novel type A h1n1 virus

A detection method and fluorescence quantitative technology, which can be used in microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc. The effect of good performance and lower detection cost

Inactive Publication Date: 2011-12-28
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limited relevant sequences published by NCBI at that time, the primers and probes published by the World Health Organization had limited specificity and sensitivity for the detection of the

Method used

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  • Fluorescent quantitative pcr detection kit and non-diagnostic detection method for novel type A h1n1 virus
  • Fluorescent quantitative pcr detection kit and non-diagnostic detection method for novel type A h1n1 virus
  • Fluorescent quantitative pcr detection kit and non-diagnostic detection method for novel type A h1n1 virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Detection of Primer Sensitivity

[0036] 1) Select the full-length gene fragment of novel influenza A (H1N1) influenza HA synthesized in vitro as a standard, and increase its viral RNA concentration from 10 -1 sequentially diluted to 10 -10 ;

[0037] 2) Negative control: normal throat swab RNA;

[0038] 3) The reaction system is prepared according to Table 3;

[0039] 4) Primer-probe combination: the primer-probe combination (YHA) designed by the present invention and the primer-probe combination (WHO-SWH1) published by the World Health Organization.

[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 The curve amplified with the primer-probe combination (WHO-SWH1) published by the World Health Organization. image 3 The curves 1-6 in the middle are the concentration dilution of the standard substance in order of 10 ...

Embodiment 2

[0045] Embodiment 2: Detection of primer characteristics

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

[0047] 2) Positive control: HA fragment RNA of new type A H1N1 influenza virus;

[0048] 3) Negative control: Normal throat swab RNA;

[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 positive control HA RNA numbered 1 (10 -6 ) 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 primer probe combination designed by the present invention has a CT value of "-" for gene amplification of common seasonal influenza H1N1, swine influenza H1N1, avian influenza H5N1 and H9N2...

Embodiment 3

[0055] Embodiment 3: detection of virus culture

[0056] 1) Detect the virus culture and screen the new type A H1N1 influenza virus;

[0057] 2) Positive control: HA fragment RNA of new type A H1N1 influenza virus;

[0058] 3) Negative control: Normal throat swab RNA;

[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 of the positive control appeared, and the rest of the samples had no amplification. The reaction was negative.

[0062] Table 6 Virus culture test results

[0063]

[0064]

[0065] The results of the data in Table 6 show that the results of the detection of virus cultures were all negative.

[0066] Result analysis:

[0067] After the amplification reaction has completed 40 cycles, the sensitivity of the primer probe combination designed by the present invention detects that the viral RNA concentration dilution is 10 -9 Compared wi...

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Abstract

The invention discloses a fluorescent quantitative PCR kit for detecting novel type A influenza virus and a non-diagnostic detection method. The kit includes primers and probes of specific sequences. The primers and probes have good detection specificity and high sensitivity, and are especially suitable for The H1N1 flu that broke out this time has no cross-reaction with bird flu, swine flu and common seasonal flu, and the cost is also greatly reduced.

Description

technical field [0001] The invention relates to a novel fluorescent quantitative PCR detection method for influenza A (H1N1) virus, as well as detection primers and specific probes. 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 situation is becoming more and more serious. In June 2009, the World Health Organization raised the alert level of the influenza pandemic to level 6. The world is at the beginning of the influenza pandemic in 2009. 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...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCY02A50/30
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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