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Outline
This study aimed to prepare a positive control for RT-qPCR detection of Porcine Reproductive and Respiratory Syndrome Virus genotype 2 （PRRSV-2） by utilizing the property of MS2 phage capsid protein to self-assemble and encapsulate exogenous nucleic acids into armored RNA， and to investigate its stability and RNase resistance. The specific target gene （conserved region of ORF7） of PRRSV-2 was inserted downstream of the PAC site in the pET28a（+）/CP-A recombinant plasmid to construct a recombinant vector. Through prokaryotic expression， ammonium sulfate precipitation， and gel filtration chromatography purification， uniform MS2 virus-like particles （VLPs） with a diameter of 20-28 nm were obtained. Benzonase nuclease digestion and RT-qPCR were used to verify its thermal stability and RNase resistance. The recombinant vector containing MS2 phage capsid protein， PAC site， and PRRSV-2 target gene was successfully constructed. After purification， armored RNA encapsulating the target gene was formed， which was resistant to RNase degradation and stable for 7 days at 37 ℃ under sterile conditions. This armored RNA can serve as a positive control for PRRSV-2 RT-qPCR detection. Compared with traditional synthetic nucleic acids， its advantages include： 1） simulating the nucleic acid state of real viruses and monitoring the entire detection process； 2） strong RNase resistance； 3） intra- and inter-batch coefficients of variation （CV） of <2% among 3 batches； 4） stable storage at 37 ℃ for 7 days. It provides a reliable quality control tool for PRRSV-2 detection.
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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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