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Multiplex PCR (Polymerase Chain Reaction) primer combination, multiplex PCR kit and detection method for rapidly detecting aflatoxin-producing bacteria

An aflatoxin and primer combination technology, applied in the field of biomedicine, can solve the problems of difficulty in producing expected results, false negatives in the system, low sensitivity, etc., and achieve the effects of comprehensive coverage, high detection rate and strong specificity

Pending Publication Date: 2022-03-04
丽水市中医院
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  • Application Information

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Problems solved by technology

[0007] However, when using multiplex PCR to identify the source of fungi, due to the competitive amplification between multiple pairs of primers, the system is prone to false negatives, non-specific amplification, and low sensitivity, which is more difficult to produce expected results than single-plex PCR; Therefore, there is a high requirement for primer specificity

Method used

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  • Multiplex PCR (Polymerase Chain Reaction) primer combination, multiplex PCR kit and detection method for rapidly detecting aflatoxin-producing bacteria
  • Multiplex PCR (Polymerase Chain Reaction) primer combination, multiplex PCR kit and detection method for rapidly detecting aflatoxin-producing bacteria
  • Multiplex PCR (Polymerase Chain Reaction) primer combination, multiplex PCR kit and detection method for rapidly detecting aflatoxin-producing bacteria

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Embodiment 1

[0055] 1. Primer synthesis

[0056] For Aspergillus versicolor A dehydrogenase gene ver-1 (the Genbank accession number of Aspergillus flavus ver-1 gene is AY510451.1, the Genbank accession number of Aspergillus parasitica ver-1 gene is M91369.1, Aspergillus oryzae ver- The Genbank accession number of 1 gene is AB007804.1), Aspergillus versicolor B gene verB (the Genbank accession number of A. The Genbank accession number of the gene is AB076804.1) and the fungal ITS sequence to design multiple PCR primers, and entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize them. The nucleotide sequences of each primer are shown in Table 1.

[0057] Table 1 Multiplex PCR primer combinations and target genes

[0058]

[0059] 2. Fungal culture and template DNA extraction

[0060] Test strains include Aspergillus flavus AS 3.3554 (H), A. parasiticus AS 3.124 (J), A. oryzae ASS.384 (Mi), A. terreus AS3 .3935(T), Aspergillus versicolor (A.versicolor) AS 3.3886(Z), Monascus ...

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Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) primer combination, a multiplex PCR kit and a detection method for rapidly detecting aflatoxin-producing bacteria. The multiplex PCR primer combination comprises a primer pair and a primer pair, wherein the primer pair is used for specifically amplifying sterigmatocystin A dehydrogenase gene ver-1, and comprises an upstream primer with a nucleotide sequence as shown in SEQ ID No.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID No.2; the primer pair is used for specifically amplifying the sterigmatocystin B gene verB and comprises an upstream primer with a nucleotide sequence as shown in SEQ ID No.3 and a downstream primer with a nucleotide sequence as shown in SEQ ID No.4; and the primer pair is used for specifically amplifying the ITS sequence of the fungus. The multiplex PCR primer combination disclosed by the invention is strong in specificity and high in sensitivity, and does not interfere with each other.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a combination of multiple PCR primers, a multiple PCR kit and a detection method for rapidly detecting aflatoxin toxin-producing bacteria. Background technique [0002] Aflatoxins are a class of toxic secondary metabolites mainly produced by Aspergillus fungi, which are highly pathogenic and carcinogenic. Aspergillus flavus and Aspergillus parasiticus are common aflatoxin-producing fungi. Aflatoxin-producing fungi usually contaminate food and Chinese medicinal materials during harvesting, processing, storage and transportation, seriously threatening human health. [0003] At present, the detection of aflatoxin is mainly through the post-column derivatization method of high performance liquid chromatography. This method can detect the content of aflatoxin more accurately, but it has the disadvantages of complicated operation and high sample volume. [0004] Identi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 张晓芹陈礼平雷后兴林娜毛佳乐
Owner 丽水市中医院
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