Fluorescent quantitative PCR primers and kit for detecting novel SADSCoV (swine acute diarrhea syndrome coronavirus)

A technology for acute diarrhea and fluorescence quantification, which is applied in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., to achieve high accuracy, efficient identification, and good repeatability

Inactive Publication Date: 2018-01-26
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] There is no report on the detection of SADS-CoV by real-time fluorescent quantitative PCR

Method used

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  • Fluorescent quantitative PCR primers and kit for detecting novel SADSCoV (swine acute diarrhea syndrome coronavirus)
  • Fluorescent quantitative PCR primers and kit for detecting novel SADSCoV (swine acute diarrhea syndrome coronavirus)
  • Fluorescent quantitative PCR primers and kit for detecting novel SADSCoV (swine acute diarrhea syndrome coronavirus)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The preparation of embodiment 1 standard positive template

[0047] 1. Extraction of total virus RNA

[0048] According to the Invitrogen company TRIZOL LS Reagent RNA extraction kit instruction manual. Add 250 μL of the aliquoted supernatant of positive disease material grinding liquid (from a pig farm in Guangdong) and 750 μL TRIZOL to a 1.5 mLeppendorf tube, mix thoroughly, and place at room temperature for 10 min; add 200 μL of chloroform, shake vigorously for 15 sec, and stand at room temperature for 5 min. Centrifuge at 12000rpm at 4°C for 15min; transfer the supernatant to a new 1.5mL eppendorf tube, add 500μL of isoamyl alcohol, mix well, place at room temperature for 10min, centrifuge at 12000rpm at 4°C for 10min; discard the supernatant, and store on ice 1000 μL of pre-cooled 70% ethanol, mix gently, wash once, centrifuge at 12,000 rpm at 4°C for 10 min; discard the supernatant, and air-dry; dissolve RNA with 20 μL of DEPC-treated triple distilled water, stor...

Embodiment 2

[0072] Example 2 Fluorescent quantitative PCR reaction condition optimization and standard curve making

[0073] The optimized fluorescent quantitative PCR reaction system is: 10 μL of 2×Premix Ex Taq (Probe qPCR), 0.8 μL of 10 μM upstream and downstream primers and probes, 1.0 μL of cDNA template, ddH 2 O supplemented to 20 μL.

[0074] The optimal fluorescent quantitative PCR reaction conditions are: 95°C for 30s; 95°C for 5s, 60°C for 30s, 40 cycles.

[0075] Determination of OD of standard recombinant positive plasmid 260nm and OD 280nm value and its ratio, calculate the plasmid concentration, and convert it into copy number, serially dilute it into 9 gradients with double distilled water 10 times (10 9 ~10 1 copies / μL). Using this as a template, a 20 μL reaction system was established, and amplified on a Bio-Rad fluorescent quantitative PCR instrument according to the optimized PCR reaction system. The obtained standard curve is shown in image 3 . As a result, the...

Embodiment 3

[0076] The sensitivity test of embodiment 3 real-time fluorescence quantitative PCR

[0077] The 10-fold serial dilution of SADS-CoV positive standard was used as a template for real-time fluorescent quantitative PCR (3.08×10 0 ~3.08×10 10 copies / μL, a total of 11 gradients, to determine their lower limit of detection. As a result, using the PMD19-T-N standard as a template, the detection limit of fluorescent quantitative PCR was 3.08×10 1 copies / μL (eg Figure 4 Shown), show that the fluorescent quantitative PCR primer and method of the present invention have high sensitivity.

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Abstract

The invention belongs to the technical field of biological detection and discloses fluorescent quantitative PCR primers and a kit for detecting novel SADSCoV (swine acute diarrhea syndrome coronavirus). The specific primers and probe are designed according to a nucleocapsid protein gene of a conserved sequence of the SADSCoV, and sequences of the primers and the probe are shown as SEQ ID NO:4-6; the SADSCoV can be amplified specifically by the primers and the probe, for real-time fluorescent quantification, fluorescent data can be acquired by monitoring specific binding condition of the probewith PCR amplified fragments in a PCR process in real time, and the SADSCoV is identified according to Cq value. The SADSCoV can be identified rapidly after PCR amplification with the method, the method is high in accuracy and specificity and good in repeatability and can identify the SADSCoV accurately, rapidly and efficiently, and popularization and application in clinical practice are facilitated.

Description

technical field [0001] The invention relates to the technical field of biological detection, more specifically, to a fluorescent quantitative PCR primer and a kit for detecting novel porcine acute diarrhea syndrome coronavirus. Background technique [0002] Coronaviruses (CoV) belong to the Coronaviridae subfamily of the Coronaviridae family and are a single-stranded positive-sense RNA virus with an envelope, which has the largest genome sequence length among known RNA viruses. Currently, there are five known porcine coronaviruses, including porcine epidemic diarrhea virus (porcine epidemicdiarrhea virus, PEDV), porcine transmissible gastroenteritis virus (transmissible gastroenteritis virus, TGEV), porcine respiratory coronavirus (porcine respiratory coronavirus, PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), and porcine deltacoronavirus (PDCoV) discovered in recent years. Among them, PEDV, TGEV, and PDCoV can cause intestinal diseases in pigs, leading to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCY02A50/30
Inventor 马静云周玲孙媛
Owner SOUTH CHINA AGRI UNIV
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