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Primer and probe for detecting drug resistance genes mecA in methicillin-resistant staphylococcus aureus

A methicillin, staphylococcus-resistant technology, applied in the field of biological detection, can solve problems such as false negatives

Inactive Publication Date: 2013-06-26
SHANGHAI PUTUO DISTRICT PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A considerable portion of the primers and / or probes are located at the sites that are prone to variation, which can easily lead to false negative results

Method used

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  • Primer and probe for detecting drug resistance genes mecA in methicillin-resistant staphylococcus aureus
  • Primer and probe for detecting drug resistance genes mecA in methicillin-resistant staphylococcus aureus
  • Primer and probe for detecting drug resistance genes mecA in methicillin-resistant staphylococcus aureus

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Experimental program
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Embodiment 1

[0028] The design and synthesis of the primers and probes used in embodiment 1

[0029] The inventor analyzed 47 pbp2a protein sequences in the NCBI database and the protein sequence in 1 clinical isolate, 23 of which were completely identical, and found the following 15 variations in the remaining 25 protein sequences, of which 246 Amino acid variation is the most common, see Table 1 for details.

[0030] Table 1

[0031]

[0032] Therefore, the inventors avoided the above variable sites when designing primers and probes. At the same time, because the A+T content of the mecA sequence is as high as 70%, the annealing temperature is low, resulting in a decrease in hybridization specificity. Therefore, the inventors used MGB probes. The 3' end of the probe is combined with MGB (minor groove binder, minor groove binder), which can be combined in the minor groove of the DNA double helix, thereby stabilizing the DNA double helix, increasing the Tm value of the probe, The hyb...

Embodiment 2

[0037] The clinical isolates MRSA (100 strains) and MSSA (50 strains) identified by routine bacterial culture were detected, and the standard strain 33591 (purchased from the American Standard Biological Collection Center, ATCC) was used as the positive control of the mecA gene, and the standard strain 25923 (purchased from American Standards Collection, ATCC) as a negative control for the mecA gene.

[0038] 1. Recover the standard strains 33591 and 25923 on the blood plate. DNA of standard strains was extracted with TIANamp Bacteria DNA Kit.

[0039] 2. Use TIANamp Bacteria DNA Kit to extract DNA from clinically isolated Staphylococcus aureus growing on blood plates.

[0040] 3. Carry out the synthesis of primers and probes according to Example 1.

[0041] Oligonucleotide name sequence target gene Nucleotide site on P 5-GGTTACGGACAAGGTGAAATACTGA-3 mecA 1552-1576 P down 5-GTGTCTTTTTAATAAGTGAGGTGCGTTA-3 mecA 1632-1658 MGB probe 5-FA...

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Abstract

The invention provides a primer and a probe for detecting drug resistance genes mecA in methicillin-resistant staphylococcus aureus (MRSA). The invention also provides a method and a detection kit for detecting drug resistance genes mecA in MRSA. According to the invention, the conserved sequence part of drug resistance genes mecA is directly detected without being affected by the mecA gene variation of strains, and a result is a gold standard of MRSA diagnosis. The specific primer and the probe provided by the invention are applicable to currently known strains containing sequence variation.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a specific primer and probe for detecting drug-resistant gene mecA in methicillin-resistant Staphylococcus aureus (methicillin resistant Staphylococcus aureus, MRSA) by PCR method. Background technique [0002] Controlling the infection and spread of MRSA in hospitals and communities is an important public health strategy worldwide. In 2007, the European Antibiotic Surveillance Network (monitoring more than 600 laboratories in 31 European countries) reported that the incidence of MRSA bacteremia per 100,000 inpatient days increased from 0.2 to 24.4. In 2005, the US Surveillance Network (monitoring 300 clinical microbiology laboratories in the United States) reported that the proportions of MRSA in Staphylococcus aureus isolated from non-ICU wards, ICU wards and outpatient clinics were 59%, 55% and 48%, respectively. The results of two meta-analyses show ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11G01N21/64
Inventor 金姝黄德魁张骥朱晴晖邹玉涵
Owner SHANGHAI PUTUO DISTRICT PEOPLES HOSPITAL
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