Comparison of five methods of extraction of Staphylococcus aureus DNA for molecular detection by PCR

Lara, Maristela Oliveira; Lucas, Thabata Coaglio; Kalapothakis, Evanguedes; Thomasini, Ronaldo Luis; Machado, Carla Jorge

doi:10.1590/0037-8682-0352-2017

Cited by 10 publications

(4 citation statements)

References 14 publications

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“…The higher male incidence is poorly understood and requires further research to identify correlated hazard agents. The result obtained from the extraction of DNA remained in accordance with the study done by Lara et al [21]. Jaffe et al [22] found that the median concentration of isolated DNA of S. aureus was 78.5 ng/µl, the difference between the results of this study and the results obtained from the current study may be explained by the use of larger quantities of bacteria in higher concentrations of DNA and kits used in DNA extraction.…”

Section: Discussionsupporting

confidence: 91%

Identification of the respiratory tract infection due to methicillin-resistant Staphylococcus aureus by TaqMan real-time PCR

Abdulsahib

2021

Medical Journal of Cell Biology

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The methicillin-resistant Staphylococcus aureus (MRSA) is a significant human pathogenic bacterium that is endemic within hospitals around the world. The identification and inspection of MRSA in clinical samples is quite helpful both in advising individual patients about the required care and in tracking these species. The goal of this study was to present a modern, faster, and more accurate diagnostic technique to operate on the real-time duplex PCR applicable to S. aureus/MRSA monitoring in Iraqi patients. For this reason, the S. aureus-specific nuc gene sequence and the mecA gene sequence were checked simultaneously. To estimate the assay efficiency, a set of six target strains, 34 non-target strains, and 296 clinical specimens were used. The findings obtained from the diagnosis of a total of 296 isolates based on phenotypic characteristics and biochemical tests showed that 146 (49.32%) were classified as individuals with respiratory tract infections of S. aureus with a total male to female ratio of 1.47, and 142 isolates demonstrated methicillin resistance. 142 MRSA isolates were investigated in the molecular analysis, all MRSA isolates had positive results for the nuc gene and 138 isolates were positive for the mecA gene. The current real-time PCR assay has 97% sensitivity, 100% specificity, and 98% accuracy. Running title: Identification of the MRSA by real time PCR

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Section: Discussionsupporting

confidence: 91%

Identification of the respiratory tract infection due to methicillin-resistant Staphylococcus aureus by TaqMan real-time PCR

Abdulsahib

2021

Medical Journal of Cell Biology

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“…A molecular analysis of phenotypically identified S. aureus isolates was performed by amplification of the Staphylococci 16s rRNA gene, the species-specific nuc gene and the mecA -resistant gene. The genomic DNA was extracted using the DNA extraction method CTAB (Cetyltrimethylammonium bromide) ( Maristela Oliveira Lara et al, 2018 ). Before molecular analysis, the extracted DNA was analyzed using gel electrophoresis (1%).…”

Section: Methodsmentioning

confidence: 99%

Methicillin-Resistant Staphylococcus aureus (MRSA) in Slaughter Houses and Meat Shops in Capital Territory of Pakistan During 2018–2019

et al. 2020

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Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a major concern in many parts of the world, including Pakistan. The aim of this study was to investigate the prevalence of MRSA in slaughterhouses and meat shops in Rawalpindi-Islamabad, Pakistan, 2018-2019. A total of 300 samples were collected: 40 from each of working area, tools (knives, hooks), butcher hands and beef, 30 from each of chicken and mutton, 20 from each of nasal and rectal swabs. S. aureus was phenotypically identified by performing gram staining and biochemical tests. 150 of the 300 samples were confirmed to be S. aureus by phenotypic identification. MRSA was identified among S. aureus positive isolates by performing disk diffusion test and by detecting S. aureus-specific genes such as 16s rRNA, nuc, mecA, spa, and coa. Out of 150 isolates 96 (63%) showed resistance to antibiotic cefoxitin, known as a potential marker for detecting MRSA. While all 150 isolates have shown complete resistance to the four antibiotics neomycin, methicillin, ciprofloxacin and tetracycline. The nuc and 16s rRNA genes were detected in all 150 S. aureus-positive isolates and 118 (79%) were confirmed to be MRSA through the detection of the mecA gene. MRSA prevalence was highest in chicken (23/30, 77%) followed by beef (25/40, 63%), mutton (15/30, 50%), knives (18/40, 45%), nasal swabs (7/20, 35%), working area (11/40, 28%), rectal swabs (5/20, 25%), hooks (7/40, 18%), and butcher hands (7/40, 18%). 50 MRSApositive isolates were chosen to identify two virulence factors (spa and coa gene). Of the 50 MRSA isolates subject to coa and spa gene typing, 27 (54%) were positive for the coa gene and 18 (36%) were positive for the spa gene, respectively. To the best of our knowledge, this was the first study on the molecular identification of MRSA in meat samples from Pakistan. High prevalence of MRSA in meat samples demand for implementation of proper hygienic practices and procedures during the slaughtering, transport and marketing of meat and meat products in order to prevent the spread of these bacteria to the human population.

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“…The PCR cycling conditions (Thermal profile) were in the order: Initial denaturation at 94˚C lasted 10 minutes; 35 cycles of amplification at 94˚C lasted for 30 seconds; annealing at 53˚C lasted for 30 seconds; extension at 72˚C lasted for a minute and finally, extension at 72˚C lasted for 5 minutes. Amplification products were analyzed on a 2.0% agarose gel electrophoresis prepared by 2 g of gel to 100 mls of TAE buffer for efficient separation [27].…”

Section: Sample Collection and Processingmentioning

confidence: 99%

Methicillin-Resistant <i>Staphylococcus aureus</i> May Also Be Resistant to Clindamycin and Vancomycin

Anejionu¹,

Oli²,

Ezeudu³

et al. 2022

JBM

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Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a global superbug widely distributed in hospitals, communities and livestock settings. This study investigated the presence and molecular characterization of MRSA co-resistance to clindamycin and vancomycin in the southeastern region of Nigeria. The susceptibility of these organisms to other selected antibiotics was also investigated. Method: Biological samples were obtained from consenting patients from three establishments in Enugu, Nigeria and cultured for isolation and purification. The pure isolates were subjected to antimicrobial susceptibility profiling using conventional antibiotics. The genomic DNAs of the pure isolates were isolated using the Promega genomic DNA purification kit while the antibiotic resistance genes (mecA) genes were identified using a multiplex polymerase chain reaction. Also, the minimum inhibitory concentration of the clindamycin and vancomycin antibiotics was determined as well as their combined activity on the MRSA isolates. Results: A large proportion (71%) of the MRSA isolates was from urine samples and then from the High Vaginal Swab (19%). All the isolates were resistant to cloxacillin while 95% were resistant to ciprofloxacin. MRSA isolates demonstrated resistance to clindamycin (with MIC of 23.

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Comparison of five methods of extraction of Staphylococcus aureus DNA for molecular detection by PCR

Abstract: Although one protocol seemed more efficient than the others, PCR was sensitive enough to allow for detection of S. aureus with all the protocols.

Cited by 10 publications

References 14 publications

Identification of the respiratory tract infection due to methicillin-resistant Staphylococcus aureus by TaqMan real-time PCR

Identification of the respiratory tract infection due to methicillin-resistant Staphylococcus aureus by TaqMan real-time PCR

Methicillin-Resistant Staphylococcus aureus (MRSA) in Slaughter Houses and Meat Shops in Capital Territory of Pakistan During 2018–2019

Methicillin-Resistant <i>Staphylococcus aureus</i> May Also Be Resistant to Clindamycin and Vancomycin

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Comparison of five methods of extraction of Staphylococcus aureus DNA for molecular detection by PCR

Abstract: Although one protocol seemed more efficient than the others, PCR was sensitive enough to allow for detection of S. aureus with all the protocols.

Cited by 10 publications

References 14 publications

Identification of the respiratory tract infection due to methicillin-resistant Staphylococcus aureus by TaqMan real-time PCR

Identification of the respiratory tract infection due to methicillin-resistant Staphylococcus aureus by TaqMan real-time PCR

Methicillin-Resistant Staphylococcus aureus (MRSA) in Slaughter Houses and Meat Shops in Capital Territory of Pakistan During 2018–2019

Methicillin-Resistant &lt;i&gt;Staphylococcus aureus&lt;/i&gt; May Also Be Resistant to Clindamycin and Vancomycin

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Methicillin-Resistant <i>Staphylococcus aureus</i> May Also Be Resistant to Clindamycin and Vancomycin