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Detection primer group for Paecilomyces hepiali Chen et Dai in ophiocordyceps sinensis culture solution, detection kit and detection method

A technology of Paecilomyces bat moth and Cordyceps sinensis, which is applied in the field of bioengineering, can solve the problems of restricting the development of Cordyceps sinensis artificial cultivation industry, the quality of bacterial liquid is difficult to guarantee, and the sampling microscope inspection misses detection, etc., and achieves low cost, simple method, and primer highly specific effect

Inactive Publication Date: 2018-05-11
INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Regarding the distinction and identification of Cordyceps sinensis and Paecilomyces spp., it can be identified by the difference in hyphae and spore morphology of microscopic examination after conventional culture, but because the content of Paecilomyces spp. , Sampling microscopic examination is not only easy to cause missed detection, but the accuracy is not high, and the operation is time-consuming, requiring operators to have professional theoretical and practical knowledge
At present, there is no standard method for detecting the quality of the culture medium of Cordyceps sinensis. It is difficult to guarantee the quality of the culture medium of Cordyceps sinensis, which greatly limits the development of the artificial cultivation industry of Cordyceps sinensis.

Method used

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  • Detection primer group for Paecilomyces hepiali Chen et Dai in ophiocordyceps sinensis culture solution, detection kit and detection method
  • Detection primer group for Paecilomyces hepiali Chen et Dai in ophiocordyceps sinensis culture solution, detection kit and detection method
  • Detection primer group for Paecilomyces hepiali Chen et Dai in ophiocordyceps sinensis culture solution, detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. The detection primer set of Paecilomyces spp. in the culture medium of Cordyceps sinensis

[0032] The detection primer set includes 2 pairs of specific primers for Paecilomyces hematoptera:

[0033] The first pair of specific primers:

[0034]Ph816F: 5'-CCTTTTGTGAACATACCTATC-3' (such as figure 1 Shown) (as shown in SEQ ID NO.1);

[0035] Ph1302R: 5'-GAGGTCAACGTTCAGAAGTC-3' (such as figure 1 Shown) (as shown in SEQ ID NO.2);

[0036] The second pair of specific primers:

[0037] Ph935F: 5'-GTATCTTCTGAATCCGCCGCA-3' (eg figure 1 Shown) (as shown in SEQ ID NO.3);

[0038] Ph1162R: 5'-CCGATTTCCCCAAAGGGAAG-3' (such as figure 1 Shown) (as shown in SEQ ID NO.4).

[0039] 2. PCR template preparation

[0040] Take the purified cultured Cordyceps sinensis Ophiocordyceps sinensis (Berk) mycelium and Paecilomyces hepiali Chen et Dai mycelia 100mg each in 2mL Microfuge tubes, grind, respectively according to the fungal CTAB genome extraction method and Fungal Genomic DN...

Embodiment 2

[0062] Embodiment 2: sensitivity analysis

[0063] Genomic DNA of purified Cordyceps sinensis and Paecilomyces hematalis was used to detect the concentration and quality of genomic DNA samples with a micro-nucleic acid spectrophotometer and electrophoresis. Then prepare 10 PCR templates according to the amount of genomic DNA of two kinds of bacteria, see Table 1, carry out nested PCR amplification (with embodiment 1 step 3) respectively with template 1~10 as template, agarose gel electrophoresis detects PCR product According to the band size, the sensitivity and accuracy of the detection primer set and detection method were confirmed.

[0064] The electrophoresis detection result of the PCR reaction product of the first round of PCR amplification shows that when the PCR template DNA (30ng of the total amount of Genomic DNA of Cordyceps sinensis and Paecilomyces thaliana) only accounts for 10 -5 (i.e. 0.0003ng), the target band of the reaction product was clear, and when the a...

Embodiment 3

[0068] Example 3: Application Analysis

[0069] In this example, 16 bottles of Cordyceps sinensis culture solution were randomly selected, and 1 mL of bacterial solution was taken from each bottle of culture solution into a 2 mL centrifuge tube, and sterile water was added, centrifuged at 8000 rpm at 10°C for 15 min, the eluate was discarded, and the sample was quickly placed in Quick-freeze in liquid nitrogen, grind, extract genomic DNA with reference to the instructions of the fungal genomic DNA extraction kit, and use 16 Cordyceps sinensis culture fluid genomic DNA as templates to carry out nested PCR amplification (same as step 3 in Example 1) to detect the Cordyceps sinensis culture fluid Presence of Paecilomyces chrysalis in . The detection result shows that in 16 Cordyceps sinensis culture fluids, Paecilomyces paecilomyces was detected in No. 5 and No. 13 bottles, and the detection primer set and detection method provided by the present invention can well detect Paecilo...

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Abstract

The invention discloses a detection primer group for Paecilomyces hepiali Chen et Dai in an ophiocordyceps sinensis culture solution, a detection kit and a detection method. Genomic DNA of the ophiocordyceps sinensis culture solution is taken as a template, two times of single-tube specific PCR (polymerase chain reaction) are performed by adopting the designed detection primer group, an amplification product is detected through agarose gel electrophoresis, and the existence condition of Paecilomyces hepiali Chen et Dai in the ophiocordyceps sinensis culture solution is judged. According to theinvention, the carrying condition of Paecilomyces hepiali Chen et Dai in the ophiocordyceps sinensis culture solution is detected through the conventional PCR, sample detection can be finished only through three steps including DNA extraction, PCR amplification and electrophoresis analysis, the method is simple to operate, easy to master and high in accuracy, and the detection kit can be furtherdeveloped with the method. The new method is provided for excluding the ophiocordyceps sinensis culture solution carrying Paecilomyces hepiali Chen et Dai, and technical support is provided for increase of infection rate of ophiocordyceps sinensis for hepialus larvae as well as stroma formation of the ophiocordyceps sinensis.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a detection primer set, a detection kit and a detection method for Paecilomyces hematophera in the culture solution of Cordyceps sinensis. Background technique: [0002] Cordyceps sinensis is a complex of dead worms and fungal subunits formed by the fungus Ophiocordyceps sinensis (Berk) infecting the larvae of the bat moth family Hepialidae. Together with ginseng and velvet antler, it is known as the three treasures of Chinese medicine. Because of its special habitat (only in alpine regions to reproduce naturally), slow growth, long cycle, and extremely limited natural resources, relevant departments have listed Cordyceps sinensis as a national second-class protected species. Artificially cultivating Cordyceps sinensis is the need to protect biological resources and the ecological environment, and it is also the need of the nation and the market. After more t...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6848C12N15/11
CPCC12Q1/6848C12Q1/6895C12Q2531/113C12Q2549/119C12Q2547/101
Inventor 刘桂清韩日畴
Owner INST OF ZOOLOGY GUANGDONG ACAD OF SCI
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