Reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae through fluorescence resonance energy transfer (FRET) and application of reagent

A technology of pathogenic bacteria and primer pairs, applied in the biological field, can solve the problems of low sensitivity and specificity, difficulty in popularization, expensive experimental equipment and reagents, etc., and achieve the effect of good detection specificity and high detection sensitivity

Inactive Publication Date: 2020-12-29
BEIJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Molecular methods are obviously superior to non-molecular methods because of their ease of use and short turnaround time, but they still have disadvantages such as slightly lower sensitivity and specificity, expensive experimental equipment and reagents, and are difficult to be widely used

Method used

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  • Reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae through fluorescence resonance energy transfer (FRET) and application of reagent
  • Reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae through fluorescence resonance energy transfer (FRET) and application of reagent
  • Reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae through fluorescence resonance energy transfer (FRET) and application of reagent

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, FRET detects Candida albicans, the establishment of Cryptococcus neoformans and Klebsiella pneumoniae method

[0033] Pathogenic bacteria were detected by PCR-FRET (fluorescence resonance energy transfer, FRET) system.

[0034] 1. Design and synthesis of primers

[0035] The specific sequence of Candida albicans (the DNA barcode of the fungus is located in ribosomal DNA (rDNA), rDNA is composed of non-transcribed region, outer transcribed spacer region, 18srDNA, ITS1, 5.8s rDNA, ITS2 and 28s rDNA. The selected The sequence comes from the ITS2 region), the specific sequence of Cryptococcus neoformans (the DNA barcode of the fungus is located in the ribosomal DNA (rDNA), and the rDNA consists of an untranscribed region, an externally transcribed spacer region, 18srDNA, ITS1, 5.8s rDNA, ITS2 and 28s rDNA. The sequences selected this time are from ITS1 to ITS2 region) and Klebsiella pneumoniae (bacterial ribosomal DNA is composed of 5s rDNA, 16s rDNA and 23s ...

Embodiment 2

[0076] Embodiment 2, the application of Candida albicans, Cryptococcus neoformans and Klebsiella pneumoniae FRET detection method

[0077] 1. Pathogen testing samples:

[0078] Non-infected samples were obtained from sterile triple distilled water. Pathogen sample: Klebsiella pneumoniae (gifted by Bayi Children’s Hospital, article: Chen Chong, Hua Shaodong, Mei Yabo, et al. Risk factors affecting the prognosis of premature infants with septic shock[J]. Zhonghua Perinatal Medicine Journal, 2018,21(12):801-807.); Cryptococcus neoformans JEC21 and H99-2 (gifted by the research group of Zhu Xudong, School of Biological Sciences, Beijing Normal University: Dong L, Xiaojiao Z, Zhongming L, et al.Cryptococcus neoformans Ca2+homeostasis requires a chloride channel / antiporter Clc1 in JEC21, but not inH99[J].Fems Yeast Research,2012(1):69-77.); Candida albicans (stored for our laboratory: Yang Qiong, Su Quanping, Wen Dezhong, et al. The immune response and vaccine research progress of...

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Abstract

The present invention discloses a reagent for detecting candida albicans, cryptococcus neoformans and klebsiella pneumoniae through fluorescence resonance energy transfer (FRET) and application of thereagent. A technical scheme is to provide a multiplex PCR primer pair composition and a single-base extension primer composition used for identifying or assisting in identifying pathogenic microorganisms, the multiplex PCR primer pair composition is composed of a primer pair A, a primer pair B and a primer pair C, and the single-base extension primer composition is composed of a primer D, a primer E and a primer F. The multiplex PCR primer pair composition and the single-base extension primer composition can specifically detect the candida albicans, cryptococcus neoformans and / or klebsiella pneumoniae in one PCR system, can accurately identify infection of any combination of the candida albicans, cryptococcus neoformans and klebsiella pneumoniae, and have sensitivity of 31.25 pg.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a reagent for detecting Candida albicans, Cryptococcus neoformans and Klebsiella pneumoniae by FRET and an application thereof. Background technique [0002] In recent years, with the wide application of high-efficiency broad-spectrum antibiotics, immunosuppressants, and anti-cancer drugs, the in-depth development of organ transplantation, catheter technology, and other interventional treatments in surgery, systemic mycoses caused by opportunistic fungi are increasing. , new pathogenic bacteria continue to appear, and the condition is becoming more and more serious. Among them, Cryptococcus neoformans (Cryptoccus neoformans) and Candida albicans (Candida Albicans) are two of the most common fungal pathogens in humans, and are likely to cause invasive infections in fungal infections. Among the bacterial infections, the infection of Klebsiella pneumoniae (Klebsiella Pneumoniae) is the...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6895C12Q1/686C12Q1/6818C12Q1/04C12N15/11C12R1/725C12R1/645C12R1/22
CPCC12Q1/689C12Q1/6895C12Q1/686C12Q1/6818
Inventor 杨琼何冰虹邱田
Owner BEIJING NORMAL UNIVERSITY
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