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Candida krusei str molecular marker and its application

A technology of Candida krusei and molecular markers, which is applied in the direction of DNA/RNA fragments, biochemical equipment and methods, and microbial measurement/inspection. Difficulty and other issues

Active Publication Date: 2021-09-07
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of pathogenic fungi, there are relatively few applications at present. One of the important reasons is that there are relatively few microsatellite sites in fungi, and it is difficult to obtain information on microsatellite sites.

Method used

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  • Candida krusei str molecular marker and its application
  • Candida krusei str molecular marker and its application
  • Candida krusei str molecular marker and its application

Examples

Experimental program
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Embodiment 1

[0026] Example 1 Screening / acquisition and primer design of Candida krusei STR loci

[0027] Based on the genome sequencing results of Candida krusei, 200 microsatellite loci were isolated from the genome sequencing data using SciRoKo software. After screening, a total of 150 pairs of microsatellite-related primers were designed by Primer premier 5.0 software, and the related primers were initially verified by e-PCR. A strain of Sporotrichum globosa was selected, and the above primers were verified by gradient PCR (while determining their appropriate annealing temperature), and 107 pairs of primers were able to amplify stably. Ten strains of Candida krusei from different sources were selected, and after genetic polymorphism detection, 40 STR loci with stable amplification and clear bands were finally obtained, as shown in Table 1.

[0028] Table 1 Microsatellite loci of Candida krusei

[0029]

[0030]

[0031]

Embodiment 2

[0032] The population genetic diversity detection of embodiment 2 candida krusei

[0033] 1. Genomic DNA extraction of Candida krusei

[0034] The yeast genomic DNA extraction kit (CW0569S) purchased from Beijing Kangwei Century Biotechnology Co., Ltd. was used to complete the genomic DNA extraction of 22 strains of Candida krusei, and the operation procedure was carried out according to the kit instructions. Twenty-one strains of Candida krusei were provided by the Laboratory Department of Peking Union Medical College Hospital.

[0035] 2. Microsatellite PCR amplification

[0036] The 21 Candida krusei samples extracted in the step are amplified by using the microsatellite site-specific primers of the present invention. The microsatellite marker-specific primer of Candida krusei was fluorescently labeled by using a fluorescent dye to label the 5' end of the forward primer with FAM.

[0037] The PCR reaction system is 20 ul, including 10 ul of 2×Premix Taq (TAKARA, Cat. No....

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Abstract

The present invention discloses Candida krusei STR molecular markers and applications thereof, which include 46 highly polymorphic microsatellite sites (SEQ ID NO: 1‑40). At the same time, a set of specific primers (SEQ ID NO: 41-120) and detection conditions that can efficiently amplify the Candida krusei population are provided. This set of microsatellite markers of Candida krusei can not only be used for genetic background analysis and molecular traceability of S. globosa, but also can be used for molecular epidemiology, population genetics and phylogenetic analysis of S. globosa .

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, in particular to STR molecular markers of candida krusei and applications thereof. Background technique [0002] Candida krusei (Candida krusei, synonym Pichia kudriavzevii, Issatchenkiaorientalis) is a clinically important pathogenic fungus worldwide. Candida krusei can cause many types of infections, including skin infections, keratitis, candidemia, and more. The pathogen is naturally resistant to the commonly used clinical antifungal drug, fluconazole. Therefore, the research on the molecular biology of Candida krusei has important scientific significance and clinical value. [0003] At present, the main molecular markers used for Candida krusei are still ITS, beta-tublin, Calmodulin, etc., but the intraspecific polymorphism of these sequences is low, which cannot satisfy the molecular traceability, population genetics and molecular prevalence of Candida krusei. The need...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 肖盟单小云龚杰范欣徐英春张建中何利华王贺
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI