Full
Outline
To establish a real-time fluorescence quantitative polymerase chain reaction (PCR) nucleic acid detection method of human parvovirus B19 and validate the method systematically.
Specific primers and probes for the highly conserved regions of the three genotypes of B19 virus were designed, recombinant vectors were constructed to prepare reference-grade plasmids, and B19 quantitative amplification standard curves were established. The linear range, accuracy, precision (repeatability and intermediate precision), detection limit, specificity, genotyping and anti-interference ability of this method were verified.
When the quantitative reference range for B19 virus was 1×104~1×108 IU·mL-1, a double logarithmic regression analysis was performed between the measured values and the theoretical values, and the regression equation R2≥0.980 0 showed good linear correlation. The deviation between the detection values of B19 standard substances at two concentration levels of sample 1 and sample 2 and the target values was≤±0.5 lg value. The CV values of B19 positive sample 1 and sample 2 for intra-batch, inter-batch precision confirmation and continuous repeated detection precision confirmation were both ≤5%. The detection limits of [STBX]NS1[STBZ] and [STBX]VP1[STBZ] genes were 57.8 and 5.78 IU·mL-1, respectively, with a detection rate ≥95%. Three genotypes of B19 virus samples can be effectively detected. The plasma of seven non B19 pathogens, including hepatitis A virus, hepatitis B virus, hepatitis C virus, human immuno-deficiency virus, human cytomegalovirus, hepatitis E virus and Treponema pallidum, was non-reactive and had good species specificity. At the same time, in the presence of seven other concurrent pathogens, the positive samples were stably detected, and the B19 nucleic acid testing method was not interfered with. This method was non-reactive to common plasma interfering substances, including hemoglobin, triglycerides and conjugated bilirubin. Meanwhile, in the presence of the above three common plasma interfering substances, B19-positive specimens were stably detected, and the B19 nucleic acid detection method was not affected.
This method has strong specificity, high sensitivity, wide linear range, good stability and reliability, and high accuracy, and can be used for the detection of human parvovirus B19 nucleic acid in plasma.
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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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