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Outline
A microdroplet digital polymerase chain reaction （ddPCR） method for the detection of fowlpox virus （FWPV） was established. In this study， a more complete ddPCR reaction system was established through the optimization of the reaction system， including the optimization of primer and probe concentrations， the optimization of annealing temperature and other steps， and reproducibility， sensitivity， and specificity experiments. In this method， the optimal primer concentration was determined to be 900 nmol·L-1， the optimal probe concentration was 200 nmol·L-1， and the results of ddPCR were optimized under the reaction condition of 55 ℃. The method showed no cross-reactivity with other common avian viruses and is highly specific. The reproducibility of the method is good， and the coefficients of variation between and within batches are less than 10%. The lowest detection limit of this ddPCR method was 2.37 copies per reaction. Using the established ddPCR detection method and the industry standard （SN/T 1226—2015） detection method for 80 samples， the coincidence rate was 100%， which was higher than the coincidence rate of 75% of the industry standard nested PCR method. In 25 artificially inoculated samples， the detection rate of ddPCR for positive samples reached 100%， while the detection rate of the nested PCR method specified in the industry standard for positive samples was 33.3%. The establishment of this method helps the relevant inspection and quarantine departments at ports to detect the FWPV in a timely manner and reduces the risk of the domestic poultry breeding industry by providing a more accurate and efficient detection method.
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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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