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Novel goose astrovirus SYBR Green dye method fluorescent quantitative PCR detection kit

A detection kit and fluorescence quantitative technology, applied in the field of poultry virus detection, can solve the problems of high technical requirements, high cost, unfavorable large-scale clinical sample detection, etc., and achieve the effects of improving detection efficiency, reducing losses, and high sensitivity

Pending Publication Date: 2019-10-22
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

qPCR mainly includes two methods of fluorescent dye labeling and TaqMan probe labeling. The probe labeling method requires high technical requirements and is expensive, which is not conducive to the detection of large-scale clinical samples.
The SYBR Green dye method does not require special probe design, is low in cost, easy to design and operate, and can be used for high-throughput detection of clinical samples. However, there is no research on the use of SYBR Green dye method to detect the new goose astrovirus. report

Method used

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  • Novel goose astrovirus SYBR Green dye method fluorescent quantitative PCR detection kit
  • Novel goose astrovirus SYBR Green dye method fluorescent quantitative PCR detection kit
  • Novel goose astrovirus SYBR Green dye method fluorescent quantitative PCR detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1: Design and synthesis of primers

[0048] The genome sequences of multiple strains of NGAstV were selected from GenBank, including NGAstV-GD strain (accession number MG934571.1), NGAstV-SDPY strain (accession number MH052598.1), NGAstV-CHN / SD01 strain (accession number MF772821.1), NGAstV-HN1G strain (accession number KY807085.1), NGAstV-FLX strain (accession number NC_034567.1), NGAstV-CXZ strain (accession number MH807626.1) and NGAstV-AHDY strain (accession number MH410610.1), using Megalign The software compared and analyzed their conserved sequences, and found that the ORF1b gene sequences of each isolate were the most conserved overall. We selected some of the conserved regions for primer design. The ORF1b gene sequence was used as a template. In order to distinguish the NGAstV-SDPY strain from other goose astroviruses, we further selected a partial sequence in the ORF1b gene sequence of the NGAstV-SDPY strain (preservation number CCTCC NO: V201808) for ...

Embodiment 2

[0052] Example 2: The composition of the kit, the optimization of experimental parameters and the investigation of specificity, sensitivity and repeatability

[0053] 1. The composition of the kit:

[0054] The kit in this embodiment is a fluorescent quantitative PCR kit for detecting NGAstV. The kit contains: the upstream primer (10 μM) shown in SEQ ID NO.1 designed in Example 1, the downstream primer (10 μM) shown in SEQ ID NO.2 designed in Example 1, standard plasmid and fluorescent quantitative PCR Reagents.

[0055] Standard plasmids are prepared by the following methods:

[0056] The NGAstV genome is used as a template and the primers shown in SEQ ID NO.1-SEQ ID NO.2 are used to obtain an amplification product, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0057] The amplified product of NGAstV was connected to the pMD18-T vector according to the operating procedures in the manual. The reaction system was: 4 μL of cDNA fragment, 1 μL of pMD18-T vector, a...

Embodiment 3

[0073] Embodiment 3: clinical application experiment

[0074] The diseased foie gras and kidney tissues with clinically suspected NGAstV infection were collected and homogenized, and the total RNA was extracted with TRIzol reagent. The RNA was reverse-transcribed into cDNA according to the operation steps using the kit, and stored at -20°C for later use.

[0075] Add 10 μL of TB Green fluorescent dye, 0.3 μL (10 μM) of upstream and downstream primers, 1 μL of cDNA template, and 8.4 μL of sterilized water into the fluorescent quantitative PCR tube, and the total reaction system is 20 μL. The reaction was carried out in a Roche LightCycle96 fluorescent quantitative PCR instrument, and the reaction conditions were: 95°C for 30s; 95°C for 5s; 60°C for 30s for 40 cycles. At the same time, standard products were added to make a standard curve.

[0076] Result Judgment Method: If the Ct value of the test sample is ≤30, and the amplification curve is a smooth "S" shape, it can be jud...

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Abstract

The invention discloses a novel goose astrovirus SYBR Green dye method fluorescent quantitative PCR detection kit. The fluorescent quantitative PCR detection kit contains primer pairs shown in SEQ IDNO.1-SEQ ID NO.2, standard plasmids and fluorescent quantitative PCR reaction reagents. The fluorescent quantitative PCR kit can detect a novel goose astrovirus, the kit is high in specificity, is only combined with the specificity of the novel goose astrovirus, and has no cross reaction with other goose-origin viruses; the detection sensitive is high, the quite good linear relation is achieved within the 1*10<1>-1*10<7> coping range of standards; novel goose astrovirus diseased goose tissue and virus carrier gooses with no clinic symptom can be effectively detected, and the detection efficiency is improved.

Description

technical field [0001] The invention relates to the technical field of poultry virus detection, in particular to a novel goose astrovirus SYBR Green dye method fluorescent quantitative PCR detection kit. Background technique [0002] Avian Astrovirus (Avastrovirus) belongs to the Astroviridae Astrovirus genus. It is a non-enveloped, single-stranded positive-sense RNA virus that can infect chickens, turkeys, ducks, geese and other birds. Avian Astroviruses include types 1, 2 and 3, and the genome length is about 6.9-7.9kb, including a 5' non-coding region (UTR) and three open reading frames (ORFs), namely ORF1a, ORF1b and ORF2,1 A 3' untranslated region (UTR) and a Poly(A) tail. ORF1a and ORF1b encode nonstructural proteins, including serine proteases, nuclear localization signals, and RNA-dependent RNA polymerase, among others. ORF2 encodes a capsid protein. The physical and chemical properties of avian astrovirus are relatively stable, and it can resist chloroform, lipid...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12Q1/6806C12R1/93
CPCC12Q1/6806C12Q1/6851C12Q1/701C12Q2531/113C12Q2563/107C12Q2521/107
Inventor 李宁刘思当蔡玉梅杨玉栋姜胜男赵君
Owner SHANDONG AGRICULTURAL UNIVERSITY
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