Multiplex PCR (polymerase chain reaction) primers, method and kit for detecting three types of toxigenic fungi polluting traditional Chinese medicinal materials

A technology for Chinese medicinal materials and fungi, applied in the field of biotechnology detection, can solve the problems of less research and complex matrix of traditional Chinese medicinal materials

Active Publication Date: 2019-02-26
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • 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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  • Multiplex PCR (polymerase chain reaction) primers, method and kit for detecting three types of toxigenic fungi polluting traditional Chinese medicinal materials
  • Multiplex PCR (polymerase chain reaction) primers, method and kit for detecting three types of toxigenic fungi polluting traditional Chinese medicinal materials
  • Multiplex PCR (polymerase chain reaction) primers, method and kit for detecting three types of toxigenic fungi polluting traditional Chinese medicinal materials

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Establishment of multiplex PCR and methodological investigation

[0079] 1. Extraction and purification method of toxin-producing bacteria DNA (modified CTAB method).

[0080] Take the concentration as 2×10 7 CFU / mL AFs-producing bacteria solution 1mL, centrifuged at 10000rpm / min for 5min, discard the supernatant. And refer to the traditional CTAB method and combined with nucleic acid purification column to extract and purify fungal genomic DNA. Specific steps are as follows:

[0081] The cultured toxin-producing bacteria were eluted from the PDA plate and collected by centrifugation. Add 0.3g of glass beads to the bacteria, add fungal lysate, shake for 15min, keep in a constant temperature water bath for 25min, place for 3min, add the same amount of chloroform: isoamyl alcohol (24:1), shake, centrifuge, take the supernatant, add 2 Mix well with / 3V / V isopropanol, transfer to a nucleic acid DNA purification column, let stand for 3 min, centrifuge at high speed fo...

Embodiment 2

[0109] Example 2 Establishment of a multiplex PCR detection system for three types of toxin-producing fungi on the surface of Chinese medicinal materials

[0110] 1. Medicinal materials

[0111] Ginseng, Panax Notoginseng, Gangmei, Bitter Almond, Peach Kernel, Morinda officinalis, Yam, Dendrobium, Rehmannia, Bergamot. Taking ginseng as the representative medicinal material, based on the multiplex PCR detection method for the three types of toxin-producing fungi established by pure bacteria, a multiplex PCR detection system for the three types of toxin-producing fungi on the surface of Chinese medicinal materials was established.

[0112] 2. Detection sensitivity of multiple PCR based on Chinese herbal medicines

[0113] Weigh 1.0g of ginseng, and use the modified CTAB method (according to the method in Example 1) to extract the medicinal material matrix, denoted as A; respectively extract three toxin-producing bacteria mixture (As3.4410 bacteria, As3.4520 bacteria and As3. 4413 bact...

Embodiment 3

[0121] Example 3 Using the established multiplex PCR detection system to detect positive medicinal materials

[0122] Detection limit test of artificial infection medicine

[0123] Take 6 blank ginseng medicinal materials, 2.0g 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, suck dry, place it in a sterile petri dish for 12-16 hours (indoor ventilation), collect them in 50mL EP tubes, add 0.1% Tween 20, Vigorously shake, take the suspension and filter, collect the filtrate (dry the filter residue for medicinal materials), centrifuge, discard the supernatant, take the bacteria and add the lysate, extract the surface of medicinal materials according to the modified CTAB method (according to the method in Example 1) Bacteria DNA, spare. As a template, a multiplex PCR detection limit test was performed. The PCR reaction conditions and system were in accordance with the three methods in Example...

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Abstract

The invention discloses multiplex PCR (polymerase chain reaction) primers, method and kit for detecting three types of toxigenic fungi polluting traditional Chinese medicinal materials. The three types of toxigenic fungi comprise aflatoxin producing fungi, sterigmatocystin producing fungi and ochratoxin producing fungi. On the basis of the multiplex PCR detection method, the multiplex PCR primerscomprise a first primer pair, a second primer pair, a third primer pair and a fourth primer pair which can be subjected to a PCR in the same reaction tube. A multiplex PCR system which directly detects the toxigenic fungi polluting the traditional Chinese medicinal materials without separation and purification, the primers have high specificity, the method has the detection limit of 10<2>-10<3> CFU / g and high detection sensitivity.

Description

Technical field [0001] The invention relates to multiple PCR primers, methods and kits for simultaneously detecting three types of toxin-producing fungi, belonging to the field of biotechnology detection, and in particular to multiple PCR primers, methods and kits for simultaneously detecting three types of toxin-producing fungi contaminated by Chinese medicinal materials. Background technique [0002] Traditional Chinese medicinal materials are affected by their own properties (containing a large amount of starch, polysaccharides, etc.). During production, processing, transportation, and storage, it is easy to contaminate various fungi and cause mildew. Not only does the value of the medicinal effect decrease, it will cause it after being taken by mistake. Poisoning. In response to the above-mentioned problem of Chinese medicinal materials being contaminated by fungi, my country has formulated a number of management practices, but there are still many gaps in pollution detection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2537/143
Inventor 徐晖詹若挺陈蔚文罗朝权王文丽鲁晓芳郑润生
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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