LAMP kit and special primer thereof for detecting streptococcus pyogenes

A technology of Streptococcus pyogenes and a kit, applied in the biological field, can solve problems such as no Streptococcus pyogenes and the like, and achieve the effect of being suitable for wide-scale popularization and application, high sensitivity and good specificity

Active Publication Date: 2015-05-20
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no LAMP kits and special primers for detecting Streptococcus pyogenes at home and abroad

Method used

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  • LAMP kit and special primer thereof for detecting streptococcus pyogenes
  • LAMP kit and special primer thereof for detecting streptococcus pyogenes
  • LAMP kit and special primer thereof for detecting streptococcus pyogenes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Primer Design for LAMP Detection of Streptococcus pyogenes

[0044] The sequence of the transcriptional regulatory gene spy1258 of Streptococcus pyogenes (GenBank number: AE006565) was retrieved from the NCBI nucleic acid database GenBank (http: / / www.ncbi.nlm.nih.gov), and homology analysis was performed by BLAST software to obtain the The specific conserved target sequence (SEQ ID NO: 1 in the sequence table) of Streptococcus pyogenes transcription regulation gene spy1258, and then according to this conserved target sequence, use the software Primer design V4 to design the primers that are used for carrying out LAMP detection to Streptococcus pyogenes, Three sets of primer combinations (code-named 36, 84 and 106) were designed, and after experimental comparison, primer combination 106 was finally selected, and the primer sequences are shown in Table 1.

[0045] Table 1 Primer combination 106 for LAMP detection of Streptococcus pyogenes

[0046]

Embodiment 2

[0047] Example 2, LAMP detection of Streptococcus pyogenes

[0048] 1. Determine the best primer combination for LAMP detection of Streptococcus pyogenes

[0049] Three sets of primer combinations (code numbers 36, 84 and 106) designed in Example 1 were used to carry out LAMP detection on Streptococcus pyogenes (from the Infectious Disease Control Center of the Chinese People's Liberation Army Institute for Disease Control and Prevention) to obtain the best primer combination, The specific method is as follows:

[0050] 1) LAMP amplification was carried out under the guidance of the above three sets of primer combinations respectively. The 25 μl LAMP reaction system included: Streptococcus pyogenes genomic DNA (Promega Genomic DNA Purification Kit A1125 to extract the nucleic acid in the sample to be tested) 2μl, 20mM Tris HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 0.1% Tween20, 0.8M betaine (betaine), 8mM MgSO 4 , 1.4mM dNTP each, 8U Bst DNA polymerase (purchased fr...

Embodiment 3

[0061] Example 3, Specificity and Sensitivity Detection of the LAMP Detection Method of Streptococcus pyogenes

[0062] 1. Specific detection of the LAMP detection method for Streptococcus pyogenes

[0063] Acinetobacter baumannii, Acinetobacter ruckeri, Bordetella pertussis, Corynebacterium diphtheriae, enteroinvasive Escherichia coli, enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, Klebsiella pneumoniae, tuberculosis Mycobacterium, Neisseria meningitidis group A, Neisseria meningitidis group B, Neisseria meningitidis group C, Neisseria meningitidis, Pseudomonas aeruginosa, Salmonella enteritidis serotype Paratyphi, Salmonella enteritidis , Shigella flexneri, Shigella sonneri, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Streptococcus pneumoniae, Vibrio sarcoides, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia enterocolitica , Yersinia pestis, Streptococcus pyogenes (the above bacterial strains all come from the Infectious Disease Control ...

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Abstract

The invention discloses an LAMP kit and a special primer thereof for detecting streptococcus pyogenes. The LAMP primer for detecting streptococcus pyogenes is designed according to a specific conservative target sequence of a streptococcus pyogenes transcriptional modulatory gene spy1258, and comprises a combination of outer primers F3 and B3, inner primers FIP and BIP and a loop primer LF. By utilizing the LAMP kit, streptococcus pyogenes in pure strains, phlegm, cerebrospinal fluid, other secretions and other samples can be qualitatively detected quickly, conveniently, efficiently, specifically and sensitively under an isothermal condition without complex instrument, and a new technical platform is provided to detection of streptococcus pyogenes.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the molecular biology of bacteria, in particular to a LAMP kit for Streptococcus pyogenes and its special primer and its application in detecting Streptococcus pyogenes. Background technique [0002] Current Situation of Clinical Infection and Treatment of Streptococcus pyogenes [0003] Streptococcus pyogenes (Group A streptococcus, GAS, defined as Group A according to different antigenic structures), β-hemolysis (meaning that it can completely hemolyze sheep blood cell agar), is an important pathogen of the human body. One of the pathogenic bacteria, can cause many kinds of diseases, the infectious diseases caused mainly include acute pharyngitis, acute tonsillitis, can also cause lung infection, scarlet fever, skin and soft tissue infection, and can cause systemic infection. Streptococcus pyogenes is also an indirect cause of allergic diseases such as rheumatic fever and acute glome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/46
CPCC12Q1/6844C12Q1/689C12Q2531/119
Inventor 袁静赵向娜贺晓明李环黄思妺刘威
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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