阅读说明：本技术 一种膨大弯颈霉菌的引物、探针以及鉴定方法 (Primer, probe and identification method for torticollis turgida ) 是由 张萍 魏锋 马双成 崔生辉 任秀 康帅 陆兔林 于 2021-09-29 设计创作，主要内容包括：本发明公开了一种膨大弯颈霉菌的引物、探针以及鉴定方法,包括所述特异性引物5’CTCTTGTTTAACCATAGTGGCA 3’和5’CTGCAATTCACATTACTTATCGC 3’,所述探针为5’TCAACAACGGATCTCTTGGCTCTG 3’。本发明设计膨大弯颈霉菌特异性引物和探针,优选后建立实时荧光PCR鉴别方法,从分子水平解决了发酵虫草类菌粉及制剂的鉴别难题。(The invention discloses a primer, a probe and an identification method of torticola turcicola, wherein the primer, the probe and the identification method comprise a specific primer 5 'CTCTTGTTTAACCATAGTGGCA 3' and a specific primer 5 'CTGCAATTCACATTACTTATCGC 3', and the probe is 5 'TCAACAACGGATCTCTTGGCTCTG 3'. The invention designs the specific primers and probes of the torticola stricta, establishes a real-time fluorescent PCR identification method after optimization, and solves the identification problem of fermented cordyceps sinensis bacteria powder and preparations from the molecular level.)

1. A primer for the bent neck mold expanding, which is characterized in that the specific primers are 5 'CTCTTGTTTAACCATAGTGGCA 3' and 5 'CTGCAATTCACATTACTTATCGC 3'.

2. A probe for the fungus Campylobacter turgidus, wherein said probe is 5 'TCAACAACGGATCTCTTGGCTCTG 3'.

3. Use of the primers and probes as claimed in any one of claims 1 to 2 in the preparation of a kit for detecting torticola turcz.

4. A kit comprising the primer and the probe according to any one of claims 1 to 2.

5. Use of the kit of claim 4 for detecting Tolypocladium turcicola.

6. The method for identifying the torticola expanse is characterized in that the total PCR reaction system of the method for identifying the torticola expanse is 25 mu L, and the reaction system comprises 2.5 mu L of 10 Xbuffer Buffer solution, 1.0 mu L of dNTP (10mmol/L), 0.2 mu L of each primer (50 mu mol/L), 0.2 mu L of probe, 0.15 mu L of Taq DNA polymerase (5U/mu L), 2 mu L of template and 18.75 mu L of sterile ultrapure water.

7. The method for identifying torticola turgida as claimed in claim 6, wherein the PCR reaction is pre-denatured at 95 ℃ for 5 minutes, and the reaction is cycled for 40 times, wherein the amplification curve is obtained at 95 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 60 seconds.

Technical Field

The invention belongs to the technical field of strain identification, and particularly relates to a primer, a probe and an identification method of torticollis.

Background

The cordyceps sinensis, as a traditional rare Chinese medicinal material, has been produced in China for thousands of years, is mainly produced in Qinghai-Tibet plateau and a transverse mountain area thereof, has the effects of tonifying lung and kidney, stopping bleeding and reducing phlegm, and relieving cough and asthma, and is called as 'three treasures of China' together with ginseng and pilose antler.

Because natural cordyceps sinensis resources are increasingly deficient and artificially cultured cordyceps sinensis is difficult to popularize in the market, the search for a cultivable substitute is urgent. From the 70 s of the last century, scientists in China have made efforts to separate various strains from wild cordyceps sinensis and artificially culture the strains into various fermented cordyceps sinensis mycelia by a fermentation technology, so that medicines with similar medicinal effects to natural cordyceps sinensis are successfully developed, and various cordyceps sinensis fermented products are successively appeared until the 80-90 s of the last century.

At present, the related research on the fermented cordyceps preparation mostly focuses on the aspects of chemical components, pharmacological action and the like of the product, and the research on the strains and the strain specificity of the cordyceps fermented product is less. The raw material of the product is zymophyte powder, the zymophyte powder is obtained by submerged fermentation culture of strains extracted and separated from cordyceps sinensis, and different strains are fermented into different bacteria powder, so that different fermented cordyceps sinensis preparations are produced. Therefore, the extracted and separated strains are the source, and the purity of the strains directly influences the quality of the fermented cordyceps preparation. In the process of subculturing and fermenting the strains, how to ensure the stability of the strains in the production period without strain variation is the foundation of ensuring the product quality, and the reports of relevant documents at home and abroad are not seen at present.

Mortierella (Mortierella SP) was first reported in 1979 to be isolated from stroma and sclerotia of fresh samples of natural Cordyceps collected from Wenchuan county, Sichuan province for a number of times. For many years, cordyceps sinensis Mortierella SP powder as a raw material medicine of Zhilingjiao capsules is considered to be a mycelium of Mortierella SP artificially cultured and fermented, and no literature report is available on molecular identification of the strain. The identification of the strain is carried out by DNA sequencing comparison, and the database comparison result shows that the strain is Tolypocladium inflatum (gb # AB255606, AB103381, AB044645 and KU182500) of the torticollis but not the strain name of the identification thereof, which indicates that the strain name of the strain powder is wrong. The strain of Tolypocladium giganteum has been reported as a model species of Tolypocladium, and how the relationship between the strain and Cordyceps sinensis is yet to be studied. The identification of the strain is mainly microscopic shape identification, and a special molecular identification method is lacked. Therefore, a primer, a probe and an identification method for the torticollis are provided.

Disclosure of Invention

The invention provides a primer and a probe for identifying fungus sources of preparations and raw material medicines, namely the torticola turcicola, from a molecular level and an accurate and exclusive identification method, so that the specificity and stability of strains and raw material medicines to preparations are ensured, namely, the strains are not mutated in the production passage process, and the stable and controllable quality of the strains, the raw material medicines and the preparations is further ensured.

The primer of the bent neck mold expanding fungus has the specific primer of 5 'CTCTTGTTTAACCATAGTGGCA 3' and 5 'CTGCAATTCACATTACTTATCGC 3'.

A probe for the fungus Campylobacter turgidus, said probe being 5 'TCAACAACGGATCTCTTGGCTCTG 3'.

The invention provides application of the primer and the probe in preparation of a kit for detecting torticola turcicola.

The invention provides a kit of the primer and the probe.

The invention provides an application of a kit for detecting torticollis turgida.

The method for identifying the torticola expanse is characterized in that the total PCR reaction system of the method for identifying the torticola expanse is 25 mu L, the reaction system comprises 2.5 mu L of 10 xBuffer Buffer solution, 1.0 mu L of dNTP (10mmol/L), 0.2 mu L of each primer (50 mu mol/L), 0.2 mu L of probe, 0.15 mu L of Taq DNA polymerase (5U/mu L), 2 mu L of template and 18.75 mu L of sterile ultrapure water.

Further, the PCR reaction was pre-denatured at 95 ℃ for 5 minutes, and the reaction was cycled 40 times, wherein 95 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 60 seconds, to obtain an amplification curve.

The invention has the beneficial effects that:

the invention designs specific primers and probes, establishes a real-time fluorescent PCR identification method for various samples such as the torticola, cordyceps sinensis tunicospora powder, Zhiling capsules and the like after optimization, and solves the problem of identifying the fungus strains of the fermented cordyceps sinensis fungus powder and preparation from the molecular level.

Drawings

FIG. 1 is a gel electrophoresis diagram of a specific primer amplification sample of a crude drug of Cordyceps sinensis powder of Ling Capsule;

FIG. 2 shows the real-time fluorescence PCR amplification curve of the powder of Campylobacter tropis and Cordyceps sinensis.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The method comprises the following steps:

taking an ITS sequence of a representative sample cordyceps sinensis fungal powder (identifying the fungal to be Tolypocladium infaltum) through sequence comparison, carrying out on-line primer and probe design by using an IDTDNA website, comparing the designed primer and probe with a sample sequence, removing a common primer and probe, synthesizing the rest primers and probes, and carrying out screening and optimization.

Screening and optimizing primer

And (3) screening and optimizing the designed primers one by adopting a PCR gel electrophoresis method, and preferably selecting a pair of primers and probe sequences with better specificity.

[ identification ]

Real-time fluorescent PCR method

About 20mg of template DNA extraction sample powder is placed in a 2mL sterile centrifuge tube, a proper amount of sterilized glass beads are added, the sample powder is ground on a high-speed ball mill with the frequency of 30 Hz/time, 1 min/time and 6 times in total, 1500 mu L of buffer solution AP and 4 mu L of RNase solution are added, vortex oscillation is carried out, DNA is extracted according to the method of the plant genome extraction kit, 150 mu L of eluent is obtained and is used as a test solution, the concentration and the purity are measured, and the sample solution is placed in a refrigerator at 4 ℃ for standby. Another control medicinal material of the cordyceps sinensis fungal powder is prepared into a control medicinal material template DNA solution by the same method, and a blank solution is extracted.

RT PCR reaction specific primers: 5 'CTCTTGTTTAACCATAGTGGCA 3' and 5 'CTGCAATTCACATTACTTATCGC 3';

a probe for the enlarged torticollis fungus: 5 'TCAACAACGGATCTCTTGGCTCTG 3'.

And (3) PCR reaction system: the reaction was carried out in 200. mu.L centrifuge tubes and the total volume of the reaction was 25. mu.L.

The reaction system included 2.5. mu.L of 10 XBuffer Buffer, 1.0. mu.L of dNTP (10mmol/L), 0.2. mu.L of each primer (50. mu. mol/L), 0.2. mu.L of probe, 0.15. mu.L of Taq DNA polymerase (5U/. mu.L), 2. mu.L of template, and 18.75. mu.L of sterile ultrapure water. Placing the centrifuge tube in a real-time fluorescent quantitative PCR instrument, wherein the PCR reaction parameters are as follows: pre-denaturation at 95 ℃ for 5 min, and cycling reaction 40 times (30 sec at 95 ℃, 30 sec at 60 ℃ and 60 sec at 72 ℃) to obtain an amplification curve.

FIG. 1 shows a gel electrophoresis chart of a specific primer amplification sample of the crude drug of the Cordyceps sinensis powder of the Ling Capsule; FIG. 2 shows the real-time fluorescence PCR amplification curve of the powder of Campylobacter tropis and Cordyceps sinensis.

Result judgment

The positive sample shows a peak before Ct 32, a positive amplification curve is shown, the control medicinal material also shows a peak before Ct 32, and the blank solution does not show a peak.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and the inventive concept within the technical scope of the present invention.

Sequence listing

<110> institute for testing and examining Chinese food and drug

<120> primer, probe and identification method for torticollis

<141> 2021-09-28

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 469

<212> DNA

<213> Zygomyces expansus (Tolypocladium inflatum)

<400> 2

ccgagttatc aactcccaaa cccctgtgaa catacccaac gttgcttcgg cgggaccgcc 60

ccggcgcctc ggcgtcccgg aaccaggcgc ccgccggagg acccaaactc ttgtttaacc 120

atagtggcat attctgagtc tcacaagaaa aatgaatcaa aactttcaac aacggatctc 180

ttggctctgg catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat 240

tcagtgaatc atcgaatctt tgaacgcaca ttgcgcccgc cagtattctg gcgggcatgc 300

ctgttcgagc gtcatttcaa ccctcaagcc ccagcggctt ggtgttgggg accggccccg 360

gccgcccccc aaatgcagtg gcgacctcgc cgcagcctcc cctgcgtagt agcacaactc 420

gcaccggagc gcggagacgg tcacgccgta aaacgcccaa cttctcaga 469