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Fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) reagent kit for detecting novel chicken reoviruses and application of fluorescent quantitative RT-PCR reagent kit

A reovirus and fluorescence quantitative technology is applied in the field of avian virus detection, which can solve the problems that the new chicken reovirus cannot be detected, the new chicken reovirus has not been detected, and cannot be quantified, and achieves high sensitivity, Loss-reducing, specific effects

Active Publication Date: 2018-08-17
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.
There have been reports on the detection of reovirus in cattle by fluorescent quantitative PCR method, but because the new chicken reovirus strain was reported for the first time, the diagnostic strategy of reovirus for mammals and other birds cannot be used for this new type of chicken reovirus. Detection of chicken reovirus, so far there is no effective method for detecting this new type of chicken reovirus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1: the design of fluorescence quantitative PCR primer and probe

[0056] According to the second-generation sequencing technology, the whole genome sequence of the new chicken reovirus (preservation number is CCTCC NO: V201817) was obtained. In the whole genome sequence, L1, L2, L3, M1, M2, M3, S1, S2, S3, S4 are The sequences of the 10 gene fragments are respectively shown in SEQ ID NO.4-SEQ ID NO.13.

[0057] Design specific primer sequences and fluorescent probe sequences for the sequence conserved region of the M1 gene, the sequence design is as follows:

[0058] Forward primer: M1-F: 5'-ATGGCCTATCTAGCCACACCTG-3'; (SEQ ID NO.1)

[0059] Reverse primer: M1-R; 5'-CAACGTGATAGCATCAATAGTAC-3'; (SEQ ID NO.2)

[0060] Fluorescent probe: 5'-FAM-TGCTAGGAGTCGGTTCTCGCA-BHQ1-3'; (SEQ ID NO.3)

[0061] The 5' end of the fluorescent probe is labeled with a fluorescent reporter group FAM, the 3' end is labeled with a fluorescent quencher group BHQ1, and the length of...

Embodiment 2

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

[0063] (1) Extraction of viral RNA:

[0064] Extract the total RNA of the sample to be tested using the classic Trizol method or a commercial kit;

[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 kit, add 2×One Step RT-PCR BufferⅢ~10μl, 1×TaKaRa Ex Taq HS(5U / μl)~0.4μl, PrimeScript RT EnzymeMixⅡ~0.4μl, PCR Forward Primer (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) ~ 2 μl, 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, 95°C for 10sec, Reps: 1; 95°C for 5sec, 60°C for 34sec, Reps: 4...

Embodiment 3

[0073] Example 3: The composition of the kit, the optimization of experimental parameters and the investigation of specificity, sensitivity and repeatability

[0074] 1. The composition of the kit:

[0075]The kit in this example is a fluorescent quantitative PCR kit, which is used to detect a new type of chicken reovirus (preservation number is CCTCC NO: V201817). 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.1, a standard and a fluorescent quantitative PCR reaction reagent.

[0076] Standards were prepared as follows:

[0077] Use the primers shown in SEQ ID NO.1-SEQ ID NO.2 to amplify the genome of chicken reovirus with the preservation number CCTCC NO: V201817 to obtain a 93bp amplification product, and connect the amplification product to pMD18-T On the ve...

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PUM

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Abstract

The invention discloses a fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) reagent kit for detecting novel chicken reoviruses and application of the fluorescent quantitative RT-PCR reagent kit. The fluorescent quantitative RT-PCR reagent kit contains primers shown as SEQ ID NO.1-SEQ ID NO.2, probes shown as SEQ ID NO.3, standard substances and fluorescent quantitative PCR reagents. The fluorescent quantitative RT-PCR reagent kit and the application have the advantages that the novel chicken reoviruses can be detected by the fluorescent quantitative RT-PCR reagent kit, and the fluorescent quantitative RT-PCR reagent kit is high in specificity and only can be specifically bound with the novel chicken reoviruses without cross reaction with other chicken-derived viruses; the fluorescent quantitative RT-PCR reagent kit is high in detection sensitivity, excellent linear relations are available in the 10<1>-10<9> copy ranges of the standard substances, the detection range can reach 9 orders of magnitude, tissues of sick chicken with the novel chicken reoviruses and virus carrying chicken without clinical symptoms can be efficiently detected by the fluorescent quantitative RT-PCR reagent kit, and the detection efficiency can be improved.

Description

technical field [0001] The invention relates to the technical field of poultry virus detection, in particular to a fluorescent quantitative RT-PCR kit for detecting novel chicken reovirus and its application. Background technique [0002] Avian orthoreovirus (ARV) belongs to the family Reoviridae and the genus Orthoreovirus. It is a pathogen that causes viral arthritis in chickens. It mainly infects broiler chickens, and chickens with both meat and eggs are also infected. ARV mainly invades the hock joints, toe joints, tibiometatarsal joints and tendons of chickens, causing joint synovitis or tenosynovitis. Diseased chickens usually show symptoms of lameness, squatting, reluctance to walk, loss of appetite or even extinction, resulting in low feed conversion rate, increased number of dead chickens, reduced production efficiency, and caused serious economic losses to the poultry industry. Since the disease was first reported in my country in the mid-1980s, the infection of A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2545/114C12Q2563/107C12Q2531/113
Inventor 刁有祥唐熠姜晓宁
Owner SHANDONG AGRICULTURAL UNIVERSITY
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