Unlock instant, AI-driven research and patent intelligence for your innovation.

Four-channel fluorogenic quantitative PCR detection method and kit for combined detection of avian influenza H7N9 and H9N2

A fluorescent quantitative and combined detection technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve difficult problems, achieve high accuracy, avoid missed diagnosis and misdiagnosis, sensitivity and specificity enhanced effect

Active Publication Date: 2017-02-22
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
View PDF9 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with conventional PCR, fluorescent quantitative PCR has advantages in sensitivity, specificity, and speed. However, real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. It can only detect up to 5 targets, and the success of the experiment is extremely difficult. Big

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Four-channel fluorogenic quantitative PCR detection method and kit for combined detection of avian influenza H7N9 and H9N2
  • Four-channel fluorogenic quantitative PCR detection method and kit for combined detection of avian influenza H7N9 and H9N2
  • Four-channel fluorogenic quantitative PCR detection method and kit for combined detection of avian influenza H7N9 and H9N2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1. Design of specific primers and probes

[0051] According to the nucleic acid sequences of H1-H16 and N1-N9 of avian influenza virus (Avian influenza virus), primers and probes (synthesized at Life Technology Company) for detection of avian influenza virus H7N9 and H9N2 genes were designed. Its sequence is as follows:

[0052]

[0053] 2. Specimen collection and pretreatment

[0054] The applicable specimen types of the method and the kit include poultry tissue, serum, throat swab, excreta, etc. Tissue samples: Take about 100-200 mg of tissue under sterile conditions, put it into a 1.5ml clean EP tube, and store it for testing. Serum: Take 3-5ml of venous blood from the tested bird with a disposable sterile syringe, keep the serum, and save it for testing. Pharyngeal and cloacal swabs: Use a cotton swab to collect secretions from the pharynx or cloacal cavity, put the swab into a centrifuge tube filled with 1.0ml PBS, and send it for inspection immediately. Sec...

Embodiment 2

[0093] Embodiment 2 Sensitivity experiment

[0094] figure 1 It is a sensitivity experiment for the detection of avian influenza virus H7 gene in a four-channel fluorescent PCR system, from left to right is 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 Amplification results of standards in cp / μl. It can be seen from the figure that the sensitivity of H7 gene detection is 1×10 2 cp / μl.

[0095] figure 2 It is a sensitivity experiment for the detection of avian influenza virus N9 gene in a four-channel fluorescent PCR system, from left to right is 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 Amplification results of standards in cp / μl. It can be seen from the figure that the sensitivity of N9 gene detection is 1×10 2 cp / μl.

[0096] image 3 It is a sensitivity experiment for the detection of avian influenza virus H9 gene in a four-channel fluorescent PCR system, from left to right is 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1Amplif...

Embodiment 3

[0098] Embodiment 3 specificity experiment

[0099] Figure 5 It is the specific experimental result of the detection of avian influenza virus H7 gene in the four-channel fluorescent PCR system. The detection of H7 antigen, H7N9 cotton swab specimens, and H7N7 cotton swab samples of Harmony Research Institute was positive, and the detection of Harmony Research H5N1 antigen, H5N1 vaccine, H9N2 cotton swab samples, Guangzhou Yongshun NDV vaccine and negative quality control products were all negative.

[0100] Image 6 It is the specific experimental result of the detection of avian influenza virus N9 gene in the four-channel fluorescent PCR system. The detection of H7N9 cotton swab specimens is positive, and the detection of H7N7 cotton swab specimens, Harmony Research H5N1 antigen, Huanong H5N1 vaccine, and H9N2 cotton swab specimens , Guangzhou Yongshun NDV vaccine and negative quality control products were all negative.

[0101] Figure 7 It is the specific experimental ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention discloses a four-channel fluorogenic quantitative PCR detection method and fluorogenic quantitative PCR kit for combined detection of avian influenza H7N9 and H9N2. By adopting the method disclosed by the invention, genes H7 and N9 of an avian influenza virus H7N9 and genes H9 and N2 of an avian influenza virus H9N2 in a sample to be tested can be simultaneously detected. A specific amplification primer and a fluorescent probe are introduced in the method, so that the sensitivity and specificity of detection can be enhanced to a large extent to avoid the problem of low specificity of other detection methods to easily result in missed diagnosis and misdiagnose. Furthermore, the kit is obtained according to principles of the method, so that the genes H7 and N9 of the avian influenza virus H7N9 and the genes H9 and N2 of the avian influenza virus H9N2 can be conveniently and simultaneously detected, and the accuracy is high.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a four-channel fluorescent quantitative PCR detection method and a kit for jointly detecting avian influenza H7N9 and H9N2. Background technique [0002] The new human infection with H7N9 subtype virus in my country has aroused widespread concern, and the regular monitoring, early diagnosis and early warning of the virus have become extremely important tasks. Studies have shown that the H9N2 subtype avian influenza virus of avian origin provides internal genes for the new recombinant virus of the H7N9 subtype influenza. However, the mechanism of how the prevalence and evolution of chicken-derived H9N2 subtype avian influenza virus promotes the emergence of the new type of avian influenza H7N9 is still unclear. , multiple H9N2 genotypes co-existed, and the G57 genotype appeared and became more adaptable to the chicken host with the change of antibody chara...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2521/101C12Q2563/107Y02A50/30
Inventor 魏文康温肖会吕殿红翟少伦曾宪淇
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI