Fluorescent RT-PCR detection method for virulent strain of Newcastle disease virus
A technique of RT-PCR and Newcastle disease virus, which is applied in the field of fluorescent RT-PCR detection of virulent strains of Newcastle disease virus, and achieves the effects of high sensitivity, convenient operation and strong specificity
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Embodiment 1
[0019] Example 1: Clinical sample detection
[0020] The concrete steps of this embodiment are:
[0021] (1) Sample collection: Collect tissue samples and cotton swabs respectively and store them at 2-8 °C for future use. The tissue samples are brain, lung, liver and other tissues of dead or sick birds; cotton swabs are oropharyngeal and cloacal swabs. ;
[0022] (2), sample processing: each sample is processed separately
[0023] 1) Tissue sample processing: Weigh 100 mg of the tissue sample to be tested and place it in a grinder, add 0.8 mL of phosphate buffered saline (PBS; pH 7.0~ 7.4), stand at room temperature for 1-2 h or at 37 °C for 30-60 min, then centrifuge at 4 °C and 1000 rpm for 10 min to take the supernatant for later use;
[0024] 2) Cotton swab treatment: put the cotton swab in 0.8 mL of PBS containing penicillin (2000 U / mL) and streptomycin (2 mg / mL), twist it well, wring it dry, discard the swab, and let it stand at room temperature For 1-2 h or at 37 °C...
Embodiment 2
[0034] Example 2: Specific Detection of Primers and Probes
[0035] The specific process of the specific detection of primers and probes in this embodiment is as follows:
[0036] (1), RNA extraction: respectively take type I Newcastle disease virus, type II virulent Newcastle disease, type II attenuated Newcastle disease, avian paramyxovirus type 4, H5N1 subtype avian influenza virus, H9N2 subtype avian influenza virus, chicken Infectious bronchitis virus, chicken infectious laryngotracheitis virus and chicken infectious bursal disease virus, respectively extract RNA for each virus, and the RNA extraction method is the same as step (5) in Example 1;
[0037] (2) Fluorescence RT-PCR reaction: use primers CIIH-172F, CIIH-389R and probe HP-353R to perform fluorescence RT-PCR amplification on the RNA template extracted in step (1), while using double distilled water as negative In contrast, the method is the same as the step (6) of Example 1;
[0038] (3) Judgment of the result...
Embodiment 3
[0039] Example 3: Sensitivity detection of primers and probes
[0040] The specific process of the sensitivity detection of primers and probes in this embodiment is as follows:
[0041] (1) Sample preparation and RNA extraction: 7 strains of Newcastle disease virulent allantoic fluid isolated from different regions, different times, different hosts and different genotypes were selected for sensitivity testing, and RNA was extracted separately. The RNA extraction method was the same as the implementation. Example 1 step (5);
[0042] (2) Fluorescence RT-PCR reaction: use primers CIIH-172F, CIIH-389R and probe HP-353R to perform fluorescence RT-PCR amplification on the RNA template extracted in step (1), while using double distilled water as negative In contrast, the method is the same as the step (6) of Example 1;
[0043] (3) Judgment of the results: After the fluorescence RT-PCR reaction was performed with 7 strains of Newcastle disease virulent as the template, amplificati...
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