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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

Inactive Publication Date: 2019-05-17
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are relatively few studies on the multiplex PCR method for Chinese herbal medicines infected with toxin-producing fungi, and they are not yet mature. One of the reasons is that the matrix of Chinese herbal medicines is relatively complex.

Method used

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  • Multi-PCR primers, method and kit for simultaneously detecting four kinds of enterotoxigenic fungi of pollution of traditional Chinese medicinal materials
  • Multi-PCR primers, method and kit for simultaneously detecting four kinds of enterotoxigenic fungi of pollution of traditional Chinese medicinal materials
  • Multi-PCR primers, method and kit for simultaneously detecting four kinds of enterotoxigenic fungi of pollution of traditional Chinese medicinal materials

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses multi-PCR primers, method and kit for simultaneously detecting four kinds of enterotoxigenic fungi of pollution of traditional Chinese medicinal materials. The four kinds of enterotoxigenic fungi are an aflatoxin production bacterium, a sterigmatocystin production bacterium and an ochratoxin production bacterium. Based on the multi-PCR detection method, the multi-PCR primers comprise a first primer pair, a second primer pair, a third primer pair and a fourth primer pair which are subjected to the PCR reaction in the same reaction tube. The multi-PCR system capable of directly detecting the enterotoxigenic fungi of pollution of traditional Chinese medicinal materials is constructed without separation and purification, all the primers are high in specificity, the detection limit of the method is 102-103 CFU / g, and the detection sensitivity is high.

Description

technical field [0001] The invention relates to multiple PCR primers, a method and a kit for simultaneously detecting four types of toxin-producing fungi, belonging to the field of biotechnology detection, in particular to multiple PCR primers, a method and a kit for simultaneously detecting four types of toxin-producing fungi polluted by traditional Chinese medicinal materials. Background technique [0002] Affected by their own properties (containing a large amount of starch, polysaccharides, etc.), Chinese herbal medicines are easy to contaminate various fungi and cause mildew during production, processing, transportation, and storage. poisoned. In response to the above-mentioned problem of fungal contamination of traditional Chinese medicinal materials, my country has formulated a number of management regulations, but there are still many gaps in pollution detection. [0003] For example, the "Chinese Pharmacopoeia" only stipulates the detection method and limit standar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/67C12R1/66C12R1/77
Inventor 徐晖鲁晓芳罗朝权詹若挺陈蔚文王文丽郑润生
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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