Fluorogenic quantitative PCR kit for detecting novel astrovirus of geese and application of kit

A fluorescent quantitative and astrovirus technology, applied in the field of poultry virus detection, can solve the problems of low sensitivity, inability to quantify, and detection blanks, etc., and achieve the effects of high sensitivity, reduced losses, and efficient detection

Active Publication Date: 2018-08-10
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Molecular biology diagnostic technology mainly includes traditional polymerase chain reaction (PCR) method and fluorescent PCR method, and traditional PCR method can only be qualitative to virus infection, can not quantify, and sensitivity is also low; Fluorescent quantitative PCR technology is with its high sensitivity, The advantages of fast speed and strong specificity have been widely used in gene expression level analysis, qualitative and quantitative detection of pathogens, etc.
However, due to the first report of the new goose astrovirus strain, the detection of the new goose astrovirus by fluorescent quantitative PCR is still a technical blank

Method used

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  • Fluorogenic quantitative PCR kit for detecting novel astrovirus of geese and application of kit
  • Fluorogenic quantitative PCR kit for detecting novel astrovirus of geese and application of kit
  • Fluorogenic quantitative PCR kit for detecting novel astrovirus of geese and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: the design of fluorescence quantitative PCR primer and probe

[0057] The whole genome sequence of the new goose Astrovirus (preservation number CCTCC NO: V201808) was obtained according to the next-generation sequencing technology, and the whole genome sequence is shown in SEQ ID NO.4. Specific primer sequences and fluorescent probe sequences were designed for the sequence conservation region of the ORF2 gene, and the sequence design was as follows:

[0058] Forward primer: ORF2-F: 5'-CCAGGTCAAGATACAATG-3'; (SEQ ID NO.1)

[0059] Reverse primer: ORF2-R; 5'-GTGTGTTCCAAGTGTAAA-3'; (SEQ ID NO.2)

[0060] Fluorescent probe: ORF2-tag: 5'-FAM-AGTCCTTCCACGATAGCCAACAT-BHQ1-3'; (SEQID NO.3)

[0061] The 5' end of the fluorescent probe is labeled with a fluorescent reporter group FAM, and the 3' end is labeled with a fluorescent quencher group BHQ1; the length of the amplified fragment is 83bp (shown in SEQ ID NO.5).

Embodiment 2

[0062] Example 2: Establishment of Fluorescent Quantitative PCR Detection Method

[0063] (1) Extraction of viral RNA:

[0064] Take 30 mg of suspected diseased gosling kidney and liver homogenate tissue, and use the classic Trizol method or a commercial kit to extract total RNA.

[0065] (2) Establishment of fluorescent PCR reaction system:

[0066] The one-step fluorescence quantification kit used adopts the One StepPrimerScript from Takara Company whose article number is RR064A TM RT-PCR Kit, add 2×One Step RT-PCR BufferⅢ~10μL, 1×TaKaRa Ex Taq HS(5U / μL)~0.4μL, PrimeScript RT Enzyme MixⅡ~0.4μL, PCR Forward Primer in 200μL PCR reaction tube (10μM)~0.4μL, PCR Reverse Primer(10μM)~0.4μL, Probe~0.8μL, ROX Reference Dye or DyeⅡ(50×)~0.4μL, Total RNA of the sample to be tested extracted in step (1)~2μL, Using RNase Free dH 2 O supplemented to 20μL system. Placed in ABI 7300Fast fluorescent quantitative PCR instrument for reaction, the reaction conditions were 42°C for 5min; 9...

Embodiment 3

[0074] Example 3: The composition of the kit, the optimization of experimental parameters and the investigation of specificity, sensitivity and repeatability

[0075] 1. The composition of the kit:

[0076] The kit in this example is a fluorescent quantitative PCR kit, which is used to detect the new goose astrovirus (preservation number is CCTCC NO: V201808). The kit contains: the forward primer (10 μM) shown in SEQ ID NO.1 designed in Example 1, the reverse primer (10 μM) shown in SEQ ID NO.2 designed in Example 1, and the reverse primer (10 μM) shown in SEQ ID NO.2 designed in Example 1 The probe shown in SEQ ID NO.3, the standard product and the fluorescent quantitative PCR reaction reagent.

[0077] Standards were prepared as follows:

[0078] The primers shown in SEQ ID NO.1-SEQ ID NO.2 were used to amplify the genome of the Goose Astrovirus whose preservation number was CCTCC NO: V201808, and an 83bp amplification product (shown in SEQ ID NO.5) was obtained. The ampl...

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Abstract

The invention discloses a fluorogenic quantitative PCR kit for detecting novel astrovirus of geese and application of the kit. The fluorogenic quantitative PCR kit is internally provided with a primershown as SEG ID NO.1-SEQ ID NO.2, a probe shown as SEQ ID NO.3, a standard substance and a fluorogenic quantitative PCR reaction reagent. The fluorogenic quantitative PCR kit can achieve detection onthe novel astrovirus of geese, is excellent in specificity and can only be specifically combined with the novel astrovirus of geese without any cross reaction with other goose source viruses; the detection sensitivity is high, a great linear relation can be maintained within a (4*100)-(4*109) copy range of the standard substance, the detection range can reach 10 orders of magnitude, sick goose tissue of geese carrying the novel astrovirus and virus carrier geese without clinical symptoms can be detected efficiently, 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 fluorescent quantitative PCR kit for detecting novel goose astrovirus and its application. Background technique [0002] Astrovirus (AStV) is a kind of non-encapsulated, spherical, single-stranded positive-sense RNA virus with a diameter of 28-30nm. The genome is infectious and 6.4-7.9kb long. The infection of this virus has a certain prevalence. It can cause enteritis and diarrhea in humans or animals, sometimes accompanied by vomiting and abdominal pain. Astroviridae can be divided into mammalian Astrovirus genus and avian Astrovirus genus according to the different infected hosts. Avian Astrovirus can cause a variety of diseases in poultry. vary with each other. Goose astrovirus (GAstV), which mainly affects young goslings, has been prevalent in many provinces, increasing the mortality rate of geese, and greatly endangering the healthy development of goose ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/6851C12Q1/701C12Q2600/166C12Q2531/113C12Q2561/101C12Q2545/114
Inventor 刁有祥唐熠杨晶
Owner SHANDONG AGRICULTURAL UNIVERSITY
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