Multi-PCR primers, method and kit for simultaneously detecting four kinds of enterotoxigenic fungi of pollution of traditional Chinese medicinal materials
A technology of Chinese medicinal materials and toxin production, applied in the field of biotechnology detection, can solve the problems of complex matrix of Chinese medicinal materials and less research
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Embodiment 1
[0087] Embodiment 1 establishes multiplex PCR and methodological investigation
[0088] 1. Extraction and purification method of toxin-producing bacteria DNA (improved CTAB method).
[0089] Take the concentration as 2×10 7 CFU / mL AFs-producing bacteria liquid 1mL, centrifuged at 10000rpm / min for 5min, and discarded the supernatant. And refer to the traditional CTAB method and combined with nucleic acid purification column to extract and purify the fungal genomic DNA.
[0090] Specific steps are as follows:
[0091] The cultured toxin-producing bacteria were eluted from the PDA plate and collected by centrifugation. Add 0.3 g of glass beads to the thalline, add fungal lysate, shake for 15 minutes, place in a constant temperature water bath for 25 minutes, place for 3 minutes, add an equal amount of chloroform:isoamyl alcohol (24:1), shake, centrifuge, take the supernatant, add 2 Mix well with 3V / V isopropanol, transfer to nucleic acid DNA purification column, let stand f...
Embodiment 2
[0110] Example 2 Establishment of a multiplex PCR detection system for four types of toxin-producing fungi contaminated on the surface of Chinese herbal medicines
[0111] 1. Medicinal materials
[0112] Codonopsis, lily, wolfberry, Ophiopogon japonicus, astragalus, ginseng, Panax notoginseng, Gangmei, bitter almond, peach kernel, Morinda officinalis, yam, dendrobium, rehmannia, bergamot. Taking Codonopsis pilosula as a representative medicinal material, based on the multiple PCR detection method of four types of toxin-producing fungi established by using pure bacteria, a multiplex PCR detection system for four types of toxin-producing fungi on the surface of Chinese medicinal materials was established.
[0113] 2. Sensitivity investigation of multiplex PCR detection based on Chinese herbal medicines
[0114] Weigh 1.0 g of Codonopsis pilosula, use the improved CTAB method (according to the method in Example 1) to extract the medicinal material matrix, which is recorded as ...
Embodiment 3
[0119] Embodiment 3 uses the established multiple PCR detection system to detect positive medicinal materials
[0120] Detection limit test of medicinal materials for artificial infection:
[0121]Take 6 parts of blank Codonopsis pilosula, 2.0 g each, soak them in different concentration gradients (2×10 2 ~10 7 CPU / mL) in the toxin-producing bacteria solution for 30s, take it out, blot dry, place in a sterile petri dish for 12-16 hours (ventilation is turned on in the room), collect them in 50mL EP tubes, add 0.1% Tween 20, Shake vigorously, take the suspension and filter, collect the filtrate (dry the filter residue medicinal materials), centrifuge, discard the supernatant, take the bacteria and add the lysate, and extract the surface of the medicinal materials according to the improved CTAB method (according to the method 1 in Example 1) Bacteria DNA, spare. As a template, a multiplex PCR detection limit test was performed. The PCR reaction conditions and system were i...
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