Identification method for paecilomyces cicadae strain

A technology for strains and strains of Paecilomyces cicadae, which is applied in the field of molecular biology and can solve problems such as identification of strains between species and within species with small morphological differences.

Active Publication Date: 2012-08-22
ZHEJIANG BIOASIA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing identification methods of Paecilomyces cicadae are identified through the characteristics of cell morphology, growth characteristics and physiological responses. These morphological identification methods are affected by environmental factors, and it is difficult to identify interspecies and intraspecies with small morphological differences. strains to be identified

Method used

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  • Identification method for paecilomyces cicadae strain
  • Identification method for paecilomyces cicadae strain
  • Identification method for paecilomyces cicadae strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] The stability test of embodiment 1RAPD fingerprint collection:

[0098] Test operation:

[0099] 1. conventional method extracts the DNA of Paecilomyces cicadae strain CGMCC No.3453, adopts primer S6, S10, S31, S80, OPV-14, OPW-06, OPC-10 carries out polymerase chain reaction (PCR) amplification, Then agarose electrophoresis was performed to detect the RAPD band pattern.

[0100] 2. PCR reaction conditions are as follows:

[0101] Reaction system: volume 25ul, DNA: 20-50ng, Mg 2+ : 50n mol, dNTP: 5n mol, primer: 10p mol, Taq enzyme: 1.5U (TAKARA Ex Taq), enzyme reaction buffer: 2.5ul (TAKARA Ex Taq Buffer), add double distilled water to 25ul, use a pipette Pipette evenly.

[0102] The PCR reaction conditions were 95°C for 3 minutes, followed by 95°C for 40 seconds, 56°C for 1 minute, 72°C for 2 minutes, and 40 cycles, then extended at 72°C for 5 minutes, and finally cooled to 4°C.

[0103] 1. Agarose gel electrophoresis, gel: 1.5% agarose gel; buffer: 1X TBE buffer...

Embodiment 2

[0109] Example 2, the RAPD polymorphism similarity of the DNA of Paecilomyces cicadae strain CGMCC No.3453 extracted in different batches:

[0110] Test operation:

[0111] 1. Get the DNA of Paecilomyces cicadae strain CGMCC No.3453 that different batches extract, carry out polymerase chain reaction (PCR) with primer S6 respectively, S10, S31, S33, OPV-14, OPW-06, OPC-10 ) amplification, followed by agarose electrophoresis to detect the RAPD band pattern.

[0112] 2. PCR reaction conditions are as follows:

[0113] Reaction system: volume 25ul, DNA: 20-50ng, Mg 2+ : 50n mol, dNTP: 5n mol, primer: 10p mol, Taq enzyme: 1U (TAKARA Ex Taq), enzyme reaction buffer: 2.5ul (TAKARA Ex Taq Buffer), add double distilled water to 25ul, pipette uniform.

[0114] The PCR reaction conditions were 95°C for 3 minutes, followed by 95°C for 40 seconds, 56°C for 1 minute, 72°C for 2 minutes, and 40 cycles, then extended at 72°C for 5 minutes, and finally cooled to 4°C.

[0115] 3. Agarose g...

Embodiment 3

[0121] Embodiment 3, RAPD similarity test of sample DNA of different passage numbers of the same bacterial strain:

[0122] Test operation:

[0123] 1. Extract the DNA of four samples of different passage numbers of the same Paecilomyces cicadae strain CGMCC No.3453, and use RAPD primers S6, S10, S31, OPV-14, and OPW-06 to carry out polymerase chain reaction (PCR) amplification. increase, and then perform agarose electrophoresis to detect the RAPD band pattern.

[0124] 2. PCR reaction conditions are as follows:

[0125] Reaction system: volume 25ul, DNA: 20-50ng, Mg 2+ : 50n mol, dNTP: 5n mol, primer: 10p mol, Taq enzyme: 1.5U (TAKARA Ex Taq), enzyme reaction buffer: 2.5ul (TAKARA Ex Taq Buffer), add double distilled water to 25ul, use a pipette Pipette evenly.

[0126] The PCR reaction conditions were 95°C for 3 minutes, followed by 95°C for 40 seconds, 56°C for 1 minute, 72°C for 2 minutes, and 40 cycles, then extended at 72°C for 5 minutes, and finally cooled to 4°C. ...

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Abstract

The invention relates to the technical field of molecular biology, and discloses a method for identifying a paecilomyces cicadae strain CGMCC No. 3453. The method comprises the following steps of: extracting DNA (Deoxyribonucleic Acid) of the strain to be detected; performing PCR (Polymerase Chain Reaction) amplification on the DNA of the strain to be detected by using primers S6, S10, S31, OPV-14 and OPW-06 respectively; performing agarose gel electrophoresis on the obtained amplification products respectively to obtain an RAPD (Random Amplified Polymorphic DNA) band graph of the strain to be detected; and comparing the obtained RAPD band graph of the strain to be detected with the RAPD band graph of a standard sample and judging to obtain an identification result. By using the identification method provided by the invention, the paecilomyces cicadae strain CGMCC No. 3453 can be quickly and accurately identified without being affected by environment.

Description

technical field [0001] The patent relates to the technical field of molecular biology, in particular to an identification method for Paecilomyces cicadae strains with a preservation number of CGMCC No.3453. Background technique [0002] Cicada flower, also known as insect flower, is the product of some cicada nymphs parasitized by Paecilomyces cicadae, and it is a complex of bacteria and insects. This fungus was named by Miquel in 1838 as Isaria cicadae. Since then, there have been many synonyms of the same thing. Such as Cordyceps cicadae, Isaria basili, Sphaeria sinclairii, Torrubia caespitosa, Cordyceps sinclairii, Isaria hariottii, Cordyceps sobolifera, Isaria sinclairii, Isaria mokanshawii and Isaria arbuscula, etc. [0003] The sexual stage of Paecilomyces cicadae is considered to be Cordyceps cicadae, Paecilomyces cicadae is widely distributed in nature, and Paecilomyces cicadae is rare. What is often collected is the parasitic complex of Paecilomyces cicadae and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 魏宁鲍晓妮江三多张健陈超孙长胜
Owner ZHEJIANG BIOASIA PHARMA CO LTD
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