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A RT-PCR, Newcastle disease virus technology, applied in the field of Newcastle disease virus strong and weak one-step real-time fluorescent RT-PCR detection kits
Inactive Publication Date: 2019-04-12
JILIN UNIV
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Problems solved by technology
At present, there is no one-step dual fluorescent RT-PCR kit for the detection of strong and weak Newcastle disease virus in the market
Method used
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Embodiment 1
[0032] Embodiment 1, design and synthesis of primers and TaqMan probes
[0033] According to the conserved sequence of the F gene of Newcastle disease virus in GenBank, multiple pairs of universal primers were designed using Primer Express 3.0 software. A set of strong and weak probes is used, and a pair of universal primers and two strong and weak probes are selected by analyzing the dimer and hairpin structure between the primers. (Table 1)
[0034] Table 1 Primers and TaqMan probe sequences (5'-3')
[0035]
Embodiment 2
[0036] Embodiment 2, the establishment of one-step real-time fluorescent quantitative RT-PCR detection
[0040] Inoculate 9-11-day-old SPF chicken embryos with NDV through the allantoic cavity of chicken embryos, and incubate them in a 37°C incubator, discard chicken embryos that died within 24 hours, aseptically collect the allantoic fluid of chicken embryos that died after 24 hours, and place in- Store in refrigerator at 80°C.
[0041] 2) Extraction of RNA
[0042]Take 200 μL of NDV virus allantoic fluid, add 600 μL Trizol lysate, shake slowly for 5-10 minutes, then add 600 μL phenol: chloroform: isoamyl alcohol (25:24:1) mixture, mix it upside down, and put it in ice bath for 15 minutes. Centrifuge at 13,500 r / min at 4°C for 15 minutes, transfer the supernatant to a tube, add an equal volume of isopropanol, mix ...
Embodiment example 3
[0070] Implementation case 3, assembly of detection kit
[0071] According to the research results of Examples 1 and 2, the detection kit is assembled for easy use
[0072] Solution A: PCR amplification buffer, universal primers F and R, probes V-T and L-T; the final concentration of primers F and R in solution A is 0.4 μM, and the final concentrations of probes V-T and L-T are 0.6 μM and 0.2 μM, respectively.
[0073] Solution B: NA-1+LaSota template, as a positive control;
[0074] Solution C: ddH 2 O served as a negative control.
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Abstract
The invention provides a Newcastle disease virus strong / weak virulent one-step real-time fluorescence RT-PCR detection kit including a primer group and a probe group; the primer group comprises a primer F and a primer R which respectively have base sequences shown in sequence tables SEQ.ID.NO.1 and SEQ.ID.NO.2. The probe group comprises a probe V-T and a probe L-T which respectively have base sequences shown in sequence tables SEQ.ID.NO.3 and SEQ.ID.NO.4. A 5' end of the strong-virulent probe V-T is labeled with a report fluorescent dye FAM, and a 3' end of the strong-virulent probe V-T is labeled with a quenched fluorescent dye BHQ1; a 5 'end of the weak-virulent probe L-T is labeled with a fluorescent reporter dye JOE, and a 3' end of the weak-virulent probe L-T is labeled with a quenched fluorescent dye BHQ2. The kit has the advantages of short detection time, high detection sensitivity and strong specificity on prevention and control of Newcastle disease viruses.
Description
technical field [0001] The invention relates to the development of a one-step dual fluorescent RT-PCR kit for detecting the strong and weak Newcastle disease virus, which belongs to a fast and sensitive safety detection technology for agricultural products, and provides technology for the "green industry" of poultry breeding and poultry products Guarantee; mainly used in agricultural and animal husbandry enterprises and regulatory agencies, specifically related to the one-step real-time fluorescent RT-PCR detection kit for strong and weak Newcastle disease virus. Background technique [0002] Newcastle disease (ND) is a poultry virulent, highly contagious, and fatal infectious disease caused by Newcastle disease virus (NDV). It mainly affects chickens and turkeys, and also infects other poultry and Wild fowl, ND has the characteristics of rapid spread, high morbidity and mortality, and it is also one of the important diseases that plague the poultry industry in my country. I...
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