Specific detection kit for isaria farinosa and detection method thereof

A detection kit and a specific technology, applied in the field of molecular biology, can solve problems such as complex physical and chemical properties of soil, affecting detection results, etc., and achieve high specificity and sensitivity, simple sample processing, and good specificity

Active Publication Date: 2018-05-01
CHONGQING TAIJI MEDICAL RES INST CO LTD +1
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Problems solved by technology

[0007] The detection of soil microorganisms based on the PCR method also has certain limitations, which is mainly due to the complex physical and chemical properties of the soil, which contains more humic acid, which can chelate Mg 2+ , HiFi enzyme or covalently combined with DNA, thus affecting the detection results, therefore, the pre-treatment of samples is the key to obtain good PCR detection results

Method used

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  • Specific detection kit for isaria farinosa and detection method thereof
  • Specific detection kit for isaria farinosa and detection method thereof
  • Specific detection kit for isaria farinosa and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Screening and verification of specific targets

[0054] The open reading frame (ORF) was predicted by retrieving the genome data of I. aeruginosa, and the target sequence without homology information was obtained by comparison. The high-abundance bacteria, fungi and Beauveria bassiana, Cordyceps militaris, and Paecilomyces spp. (Paecilomyces hepiali) etc. as background. 105 pairs of primers were designed, and the sequences of some primers are shown in Table 1.

[0055] Table 1. Primer pairs for detection of I.

[0056]

[0057]

[0058]

[0059] Use the designed primers to carry out PCR detection on the genomic DNA of Penicillium pointis, Penicillium grassa, Isera clover, Beauveria bassiana, Paecilomyces batalis and Cordyceps militaris. The detection system is as follows: template DNA 1μl, Tar-1F 0.5 μl, Tar-1R 0.5μl, PrimeSTAR Max Premix (2×) 12.5μl, ddH 2 O 10.5 μl, total volume 25 μl; amplification conditions are as follows: pre-denaturation a...

Embodiment 2

[0068] Embodiment 2, the establishment of the detection system of Isyria clumps in soil samples

[0069] A method for extracting nucleic acid in a soil sample, comprising the steps of:

[0070] 1) Take 500mg of soil sample and add 500mg of quartz sand, fully grind;

[0071] 2) Add 3mL of cleaning solution to the grinding sample, shake for 5min, centrifuge at room temperature (18-25°C) at 12000rpm for 5min, discard the supernatant, repeat twice, add 3mL of PBS buffer, and wash once;

[0072] 3) Add 5 mL of lysate, 500 μL of lysozyme (20 mg / mL), and 15 μL of proteinase K (20 mg / mL) to the above-mentioned washed centrifuge tube, shake for 10 min, 37 °C water bath for 30 min, centrifuge at 8000 rpm for 10 min, and transfer the supernatant to into a new 5mL centrifuge tube;

[0073] 4) Add an equal volume of phenol / chloroform / isoamyl alcohol to the supernatant, mix it upside down, centrifuge at room temperature (18-25°C) at 12000rpm for 10min, and collect the supernatant into a n...

Embodiment 3

[0075]Embodiment 3, the detection of Isyriatium trichomonas in soil

[0076] According to the method of Example 2, the total nucleic acid in the soil was extracted as a template, and Tar-1F / Tar-1R and ITS5-172 / ITS4-95 were used as primers for PCR amplification. The amplification system and amplification conditions were the same as in Example 1. At the same time, PCR amplification was carried out using the total nucleic acid extracted without cleaning solution, using PBS instead of lysate, without adding I. clavium, and without using lysate as templates. The amplification results were detected by electrophoresis, and the results were as follows: Figure 5 shown. The results show that according to the steps given in technical scheme 2, the total nucleic acid in the soil extracted after the cleaning solution and lysate treatment is used as a template, and Tar-1F / Tar-1R and ITS5-172 / ITS4-95 are used as primers for PCR Amplification can amplify the target band. Therefore, only b...

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Abstract

The invention relates to a specific detection kit for isaria farinosa and a detection method thereof. The kit comprises a Tar-1F / Tar-1R primer pair, wherein sequences of the Tar-1F / Tar-1R primer pairare shown as SEQ ID NO. 41 and SEQ ID NO. 42; the primer can be used for specifically recognizing the isaria farinosa; the invention further discloses the method for detecting isaria farinosa pollution in a soil sample; soil sample detection is realized by washing a soil sample through a washing solution, treating by utilizing a lysis solution, lysozyme and proteinase K and purifying the soil sample and detecting. According to the method provided by the invention, humic acid in soil is removed and a condition that a detection result is influenced, caused by covalent binding between humic acidchelated Mg<2+> and an HiFi enzyme or DNA (Deoxyribonucleic Acid), is prevented; the accuracy is good and the sensitivity is high; the invention further discloses a method for detecting isaria farinosa pollution in polypides and has important meanings on isaria farinosa detection in an artificial cultivation process of caterpillar fungus.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a specific detection kit for Isyrrhizae amygdala, and also relates to a method for detecting Isysmus aesculi in soil samples. Background technique [0002] Cordyceps sinensis refers to the complex of insects and fungi formed by the infection of bat moth larvae by Ophiocordyceps sinensis, which is a traditional Chinese medicinal material in my country. In recent years, with the changes in the global climate and the ecological environment of the Qinghai-Tibet Plateau and the over-harvesting of Cordyceps sinensis, the price of Cordyceps sinensis is expensive, and the resources of wild Cordyceps sinensis are on the verge of depletion. In order to protect the ecological environment and Cordyceps resources on the Qinghai-Tibet Plateau, and at the same time make Cordyceps serve human health, it is imminent to carry out large-scale artificial cultivation of Cordyceps. In the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2527/125
Inventor 秦少容李春峰卿玉玲陈仕江陈若霓鲁增辉仝超群
Owner CHONGQING TAIJI MEDICAL RES INST CO LTD
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