Real-time fluorescent PCR method for detecting mycobacterium avium and application of primer pairs

A technology of Mycobacterium avium and bacteria, which is applied in the field of real-time fluorescent PCR detection of Mycobacterium avium, can solve the problems of infrequent use, poor accuracy and complicated operation, and achieves the effects of reducing the probability of errors, high accuracy and low price

Active Publication Date: 2020-03-31
BEIJING TUBERCULOSIS & THORACIC TUMOR RES INST +1
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, traditional biochemical identification methods are not commonly used due to complex operations and poor accuracy; methods relying on homologous DNA sequence comparisons have become the "gold standard" for identification of bacterial species, but are difficult to be widely used due to high prices in clinical

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Real-time fluorescent PCR method for detecting mycobacterium avium and application of primer pairs
  • Real-time fluorescent PCR method for detecting mycobacterium avium and application of primer pairs
  • Real-time fluorescent PCR method for detecting mycobacterium avium and application of primer pairs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, strain and its accuracy analysis

[0071] 1. Strains

[0072] 21 standard strains of mycobacteria: Mycobacterium avium (ATCC25291), Mycobacterium intracellulare (ATCC13950), Mycobacterium abscessus (ATCC19977), Mycobacterium chelonis (ATCC14472), Mycobacterium kansasii (ATCC12478), Ge Mycobacterium aureus (ATCC14470), Mycobacterium fortuitum (ATCC6481), Mycobacterium phlei (ATCC11758), Mycobacterium aureus (ATCC23366), Mycobacterium scrofula (ATCC19981), Mycobacterium light yellow (ATCC43909), stigmata Mycobacterium smegmatis (ATCC19420), Mycobacterium tuberculosis (ATCC27294), Mycobacterium malmosa (ATCC29571), Mycobacterium simianum (ATCC25275), Mycobacterium suja (ATCC35799), Mycobacterium suis (ATCC33776), Mycobacterium terrae (ATCC15755), Mycobacterium flavinum (ATCC14474), Mycobacterium disidei (ATCC19340), Mycobacterium stutzeri (ATCC27962).

[0073] Five common pathogenic bacteria: Staphylococcus aureus (ATCC29213), Escherichia coli (ATCC25922), ...

Embodiment 2

[0079] Embodiment 2, the acquisition of the primer that is used to detect Mycobacterium avium

[0080] 1. Primer design, screening and preparation

[0081] A large number of sequence analyzes and comparisons were carried out to obtain several primers for identifying Mycobacterium avium. Preliminary experiments were performed on each primer to compare performances such as sensitivity, specificity, and repeatability, and finally four pairs of primers for identifying Mycobacterium avium were obtained, as shown in Table 1.

[0082] Table 1 is used to identify four pairs of primers of Mycobacterium avium

[0083]

[0084] Note: F indicates the forward primer, R indicates the reverse primer.

Embodiment 3

[0085] Embodiment 3, accuracy

[0086] Four pairs of primers in Table 1 were used to amplify the genomic DNA of the Mycobacterium avium standard strain respectively; 10 μl of the amplified product was carried out on a 2% agarose gel, and the amplified product was sequenced at the same time, and the sequencing results were used in BLAST and GenBank databases. The amplified sequences of each standard bacteria gene were compared to analyze and evaluate the accuracy of the mycobacterium standard bacteria used in this study.

[0087] The results showed that the sequencing results of the amplified products of primer pairs 1, 2, 3 and 4 were aligned with the target gene sequences in the GenBank database, and the concordances were 100%, 100%, 100%, and 99.32%, respectively. It shows that the four pairs of primers for Mycobacterium avium designed in the present invention all have higher diagnostic accuracy for Mycobacterium avium.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to view more

Abstract

The invention discloses a real-time fluorescent PCR method for detecting mycobacterium avium (MA) and an application of primer pairs. The invention provides the specific primer pairs, and establishesthe real-time fluorescent PCR method for detecting the MA based on SYBR Green I fluorescent dye. The method has high sensitivity, good specificity and good reproducibility, and has the advantages of acheaper price and easier operation compared with those of methods such as a DNA chip technology and a PCR-fingerprint; compared with a current ''gold standard'' of strain identification, the method has no significant differences for detecting the MA, and the simple, cheap, and highly accurate molecular biology method is provided for clinical identification of the MA; the method improves the workefficiency of inexperienced experimental technicians, reduces the difficulty of experimental operation, and reduces the probability of errors, so that the experimental technicians can perform experiments after being trained; and compared with other molecular biology methods, in the case of having higher diagnostic accuracy, the method provided by the invention reduces costs, reduces experimental funds, and is easier to promote clinically.

Description

technical field [0001] The invention relates to a method for detecting mycobacterium avium by real-time fluorescent PCR and its application. Background technique [0002] Studies in different regions of my country have shown that Mycobacterium avium (MA) is the main non-tuberculous mycobacterium (NTM) prevalent in my country. Mycobacterium avium is a zoonotic pathogen that can cause lung, soft tissue, lymph node, and skin infections in humans. MA infection not only lacks specificity in clinical manifestations and histopathology, but also has a significant difference in the drug resistance spectrum of Mycobacterium intracellulare. Therefore, the identification of Mycobacterium avium species is of great significance to determine the source of infection, analyze the transmission route, and formulate a reasonable treatment plan. [0003] At present, traditional biochemical identification methods are not commonly used due to complex operations and poor accuracy; methods relying...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/325
CPCC12Q1/689C12Q1/686C12Q2561/113C12Q2563/107
Inventor 刘洋王赛赛潘丽萍姜广路林婷婷李自慧贾红彦孙琦张宗德
Owner BEIJING TUBERCULOSIS & THORACIC TUMOR RES INST
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap