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Equine influenza detection kit and detection method

A technology of equine influenza and kits, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of inability to calculate the starting DNA or RNA copy number, inability to achieve accurate quantification, etc. question

Inactive Publication Date: 2009-03-25
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there is no linear relationship between the amount of the final PCR product and the amount of the initial template, the copy number of the starting DNA or RNA cannot be calculated based on the final PCR product amount, and accurate quantification cannot be achieved.

Method used

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  • Equine influenza detection kit and detection method
  • Equine influenza detection kit and detection method
  • Equine influenza detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Embodiment 1: the preparation and use of kit

[0136] 1. See Table 3 for the composition of the kit.

[0137] Table 3 Kit preparation composition

[0138] Composition (48tests / box) quantity lysate 5.0mL×6 tubes H3 and N8 Subtype Equine Influenza Virus Fluorescence

RT-PCR Single Reaction Solution Each 750μL×1 tube Fluorescence RT-PCR of H3 N8 Subtype Equine Influenza Virus

double reaction solution

750μL×1 tube Taq enzyme (5U / μL) 45μL×1 tube RT-PCR enzyme particles 1 capsule x 36 tubes DEPC water 1mL×3 tubes negative control 1mL×3 tubes positive control 1mL×3 tubes

[0139] 2. How to use the kit

[0140] 2.1 RNA extraction:

[0141] Take n 1.5ml sterilized centrifuge tubes (n = number of samples + 1 tube of negative control + 1 tube of positive control) and mark them. First add 600ul of lysate (the lysate is highly corrosive, do not touch the skin or clothes, otherwise rinse with plenty...

Embodiment 2

[0153] Embodiment 2: the sensitivity test of kit and be used for the relative quantification of virus RNA

[0154] 1. Materials:

[0155] Methods The virus strain used in the research process was the H3N8 subtype equine influenza virus A / equine / xibei / 1 / 2007 (10 -5 EID50 / 0.1ml).

[0156] 2. Method

[0157] 1) The allantoic fluid of H3N8 subtype equine influenza virus A / equine / xibei / 1 / 2007 chick embryo culture was used as 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 Fluorescence RT-PCR detection of H3 and N8 single and double H3N8 subtype equine influenza viruses were performed respectively, and the viruses of each dilution were inoculated into SPF chicken embryos, and the sensitivity of the two methods was compared.

[0158] 2) Relative quantification of viral RNA:

[0159] In order to achieve the relative quantification of the amount of viral RNA in clinical samples, we further detected the serial 10-fold dilution of viral RNA samples, per...

Embodiment 3

[0163] Embodiment 3: the specificity test of kit

[0164] 1. Materials

[0165] Table 4 Virus strains applied in the method research process

[0166]

[0167] 2. Method

[0168] Use the established single and double fluorescent RT-PCR method to detect a variety of influenza viruses (including H1, avian H3, H5, H9 subtype viruses), equine arteritis virus, and vest type 1 influenza virus to verify the method specificity.

[0169] 3. Results

[0170] Figure 5 and Figure 6 As shown, the results showed that the established method had no cross-reaction with the above viruses and had good specificity.

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Abstract

The invention relates to a horse influenza detecting reagent kit and a detecting method, belonging to the inspection and quarantine field. A group of nucleotide sequences for detecting H3N8 subtype horse influenza virus is the nucleotide sequences showed in sequence lists from SEQ ID No: 1 to SEQ ID No: 6. The method has the advantages of: (1) fastness: the detecting time is shortened from 21 days which the traditional detecting method to 4 hours; (2) sensitivity: the H3N8 subtype horse influenza virus which is gradient-diluted for 10 times is detected and the results indicate positive while diluted to 10<-7> times by a single approach and negative while diluted to 10<-5> by double approaches; (3) specificity: when the established H3N8 subtype horse influenza virus single-approach and double-approach fluorescent RT-PCR detecting method is used for detecting equine arteritis virus and other subtype influenza viruses, the result is negative and no cross-reaction is found; (4) stability: results of repeated experiments show that the established method has good stability; and (5) being not easy for contamination: the totally closed-up reaction requires no PCR post-treatment, thus being safe in operation.

Description

technical field [0001] The invention relates to a detection kit and a detection method for equine influenza, especially to the nucleotide sequence, detection kit and method for detecting the H3N8 subtype equine influenza virus using a rapid single-fold and double fluorescent RT-PCR detection method. For the accurate detection of H3N8 subtype equine influenza virus in animal tissue samples, it can also be applied to the detection of living animal samples (mainly nasal swabs), and can be used for the screening of domestic equine animal H3N8 subtype equine influenza virus. The quarantine of entry horses, etc., belongs to the field of inspection and quarantine. Background technique [0002] Equine influenza (abbreviated as equine influenza) is caused by equine influenza virus (Equine influenza, EI), which can cause acute upper respiratory tract infection in horses. The characteristic clinical symptoms are fever, dyspnea, anorexia and persistent cough; Cause abortion in mares; p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12N15/11
Inventor 高志强赖平安刘月焕柏亚铎乔彩霞周琦谷强蒲静张向东汪琳吴丹段生涛
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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