Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp

Sudhaharan, Sukanya; Vanjari, Lavanya; Neeraja, M.; Ede, Nagapriyanka; Lakshmi, V

doi:10.7860/jcdr/2015/13962.6315

Cited by 16 publications

(14 citation statements)

References 20 publications

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“…Staphylococcus aureus produced an extracellular thermostable nuclease, encoded by the nuc gene, whose activity has been used for the rapid and direct detection of Staph. aureus (Sahebnasagh et al, 2014;Sudhaharan et al, 2015). The results of this study showed that 85.7% of the Staph.…”

Section: Discussionmentioning

confidence: 56%

Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China

Dong

Zhang

Zhou

et al. 2016

Journal of Dairy Science

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Staphylococcus aureusis the leading pathogen involved inbovine mastitis, but knowledgeabout antimicrobial resistance, virulence factors, and genotypes of Staphylococcus aureus resulting in bovine mastitis in Ningxia, China, is limited. Therefore, antimicrobial susceptibility, virulence gene, and randomly amplified polymorphic DNA (RAPD) analyses of Staph. aureus were carried out. A total of 327 milk samples from cows with clinical and subclinical mastitis in 4 regions of Ningxia were used for the isolation and identification of pathogens according to phenotypic and molecular characteristics. Antimicrobial susceptibility against 22 antimicrobial agents was determined by disk diffusion. The presence of 8 virulence genes in Staph. aureus isolates was tested by PCR. Genotypes of isolates were investigated based on RAPD. Results showed that 35 isolates obtained from mastitis milk samples were identified as Staph. aureus. The isolates were resistant to sulfamethoxazole (100%), penicillin G (94.3%), ampicillin (94.3%), erythromycin (68.6%), azithromycin (68.6%), clindamycin (25.7%), amoxicillin (11.4%), and tetracycline (5.7%). All of the isolates contained one or more virulence genes with average (standard deviation) of 6.6±1.6. The most prevalent virulence genes were hlb (97.1%), followed by fnbpA, hla, coa (94.3% each), nuc (85.7%), fnbpB (80%), clfA (77.1%), and tsst-1 (40%). Nine different gene patterns were found and 3 of them were the dominant gene combinations (77.1%). Staphylococcus aureus isolates (n=35) were divided into 6 genotypes by RAPD tying, the genotypes III and VI were the most prevalent genotypes. There was greatvariation in genotypes of Staph. aureus isolates, not only among different farms, but also within the same herd in Ningxia province. The study showed a high incidence of Staph. aureus with genomic variation of resistance genes, which is matter of great concern in public and animal health in Ningxia province of China.

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Section: Discussionmentioning

confidence: 56%

Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China

Dong

Zhang

Zhou

et al. 2016

Journal of Dairy Science

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“…Various methods like culture, PCR, MIC determination, and antibiogram are applicable for the detection of MRSA, although all of the mentioned methods suffer from some drawbacks. Molecular methods like PCR and real-time PCR are considered as methods of choice, as they are sensitive, accurate, and less time-consuming in comparison with culture and biochemical methods (26,27).…”

Section: Discussionmentioning

confidence: 99%

Diagnostic Values of Multiplex Loop-Mediated Isothermal Amplification and Multiplex Polymerase Chain Reaction for Detection of Methicillin-Resistant Staphylococcus aureus

Kashani

Alvandi

Abiri

2020

Jundishapur J Microbiol

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Background: Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant pathogen in community and hospital environments and is associated with high mortality and morbidity. Both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) methods are sensitive and acceptable molecular methods for the diagnosis of infectious diseases. Objectives: This study aimed to develop detection assays for Staphylococcal mecA and spa using multiplex PCR and LAMP. Methods: Both methods were standardized, and detection limits were determined using serial dilutions of S. aureus DNA samples. Fifty-three clinical isolates of S. aureus were confirmed to the species level using biochemical tests and multiplex PCR and multiplex LAMP for the spa gene, while disk diffusion, minimum inhibitory concentration, and detection of mecA genes were used for the assessment of methicillin resistance. Results: The PCR could detect the mecA and spa genes at 1 fg/mL and 10 fg/mL of bacterial DNA, which were equal to 35 and 350 gene copy numbers, respectively. Similarly, multiplex LAMP detected the spa and mecA genes at 0.1 fg/mL and 1 fg/mL of bacterial DNA, which were equal to 3.5 and 35 genome copy numbers, respectively. According to MIC and disk diffusion methods, four (7.54%) cases were oxacillin-sensitive methicillin-resistant S. aureus, 16 isolates were methicillin-sensitive, and 37 isolates were methicillin-resistant. According to multiplex PCR, 47.75% of the isolates were mecA-positive while in multiplex LAMP, 41 (35.77%) isolates were mecA-positive. Conclusions: The sensitivity and specificity of the multiplex LAMP were higher than those of multiplex PCR and biochemical methods. Thus, we can apply the LAMP for the routine detection of MRSA.

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“…Based on the sequence information and previous report (Sudhaharan et al. 2015 ), a set of LAMP primers targeting nuc gene was chosen. At the same time, the forward and reverse PCR primers of nuc gene were also designed by Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Generally, it requires a lengthy time span of 3–5 d. Molecular biology techniques such as polymerase chain reaction (PCR), real-time PCR and DNA hybridization have been developed and become popular for pathogen identification (Sudhaharan et al. 2015 ). Although these technologies can detect low number of bacterial cells, they usually need several hours.…”

Section: Introductionmentioning

confidence: 99%

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Direct loop-mediated isothermal amplification assay for on-site detection of Staphylococcus aureus

Tian

Feng

Wang

2018

FEMS Microbiology Letters

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Staphylococcus aureus (S. aureus) is a major human pathogen that may produce a variety of toxins and cause staphylococcal food poisoning. In the present study, a direct loop-mediated isothermal amplification (LAMP) assay was developed and validated to detect S. aureus in food samples. Without prior cultural enrichment and DNA extraction steps, bacterial DNA was released by heating at 100°C for 5 min and directly subjected to LAMP assay. Using a set of LAMP primers recognizing six distinct regions of nuc gene, the developed direct LAMP assay was highly specific, and the analytical sensitivity was determined to be 7.6 × 102 CFU/mL. Moreover, with the pre-mixed LAMP reagents stored at –20°C, the entire assay should be finished within 40 min. These features greatly simplified the operating procedure and made the direct LAMP a powerful tool for rapid and on-site detection of S. aureus in food samples.

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Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp

Cited by 16 publications

References 20 publications

Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China

Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China

Diagnostic Values of Multiplex Loop-Mediated Isothermal Amplification and Multiplex Polymerase Chain Reaction for Detection of Methicillin-Resistant Staphylococcus aureus

Direct loop-mediated isothermal amplification assay for on-site detection of Staphylococcus aureus

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