Full
Outline
To realize the rapid detection of toxin-producing Aspergillus carbonarius, and to establish a dual fluorescence quantitative polymerase chain reaction (qPCR) method for detecting the polyketide synthases(PKS) gene and nonribosomal peptide synthetase (NRPS) gene of Aspergillus carbonarius.
Taking the relatively conserved gene fragments of PKS and NRPS from toxin-producing Aspergillus carbonarius as targets,specific primer-probe sets were designed and screened respectively. After optimizing the reaction conditions of dual qPCR, the standard curve of the assay was established. Subsequently, the specificity, sensitivity, and repeatability of this method were verified. Finally, this method was used to test 80 actual samples and corn, peanut and soybean samples artificially inoculated with different concentrations of toxin-producing Aspergillus carbonarius spores.
The specificity test results demonstrated that this dual qPCR assay exhibited no non-specific amplification with common toxin-producing fungi such as Aspergillus flavus and Aspergillus parasiticus, but specifically amplified the PKS and NRPS genes of toxin-producing Aspergillus carbonarius. The sensitivity assay showed that the limit of detection of this method could reach 1×102 copies/μL. And the method presented excellent repeatability, with the intra- and inter-batch coefficients of variation both less than 1%. In the simulated sample test, this dual qPCR could detect spores at a minimum concentration of 1×103 spores/mL in all 3 kinds of artificially infected samples.
The established dual fluorescence quantitative PCR has high sensitivity and accuracy, it can provide technical support for the early monitoring and control of ochratoxin A-toxin-producing Aspergillus carbonarius.
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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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