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Outline
This study aimed to construct a safe and effective Newcastle disease virus （NDV） pseudovirus for the quality control of NDV nucleic acid detection. The P gene of NDV was amplified by RT-PCR and linked to the lentiviral expression vector PLV-Puro to construct Puro-NDV/P. Puro-NDV/P plasmids and two helper plasmids were extracted， and a total of 293T cells were transfected， and pseudovirus particles containing NDV P genes were successfully constructed. The constructed pseudovirus particles were properly diluted and lyophilized to prepare RNA-positive quality controls， and calibrated， uniformity and stability tested. A Taqman fluorescence quantitative PCR detection method was established by successfully constructing NDV P gene pseudovirus. The results showed that 15 positive quality control products were randomly selected for fluorescence RT-PCR detection， and the Ct value coefficient of variation （CV） was 0.90%， which was good uniformity. The copy number of the prepared positive quality control products was 6.58×102 copies·μL-1. The quality controls were stored at 4 ℃， 25 ℃ for short-term storage （1 week）， and -20 ℃ for a long time （6 months）， and the Ct value was detected by fluorescence RT-PCR without significant change， and the stability was good. A real-time fluorescence RT-PCR detection method based on NDV P gene pseudovirus was established. The NDV pseudovirus prepared in this test has the advantages of good uniformity and stability， and can be used for the research and development， evaluation and quality control of NDV nucleic acid detection kits， providing a reliable tool for the standardized detection of NDV.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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