RESUMEN
COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 °C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.
Asunto(s)
Prueba de COVID-19/métodos , COVID-19/diagnóstico , Colorimetría/métodos , Tamizaje Masivo/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , SARS-CoV-2/genética , COVID-19/genética , COVID-19/virología , Humanos , ARN Viral/análisis , Transcripción Reversa , SARS-CoV-2/aislamiento & purificaciónRESUMEN
This study reports loop-mediated isothermal amplification (LAMP) for rapid detection of methicillin-resistant Staphylococcus aureus from direct clinical specimens. Four primers including outer and inner primers were specifically designed on the two target sequences-femB to identify S. aureus and mecA to identify antibiotic-resistant gene. Reference strains including various species of gram-positive/gram-negative isolates were used to evaluate and optimize LAMP assays. The optimum LAMP condition was found at 63°C within 70 min assay time (include hybridization with FITC probe for 5 min and further 5 min for reading the results on the lateral flow dipstick). The detection limits of LAMP for mecA was 10 pg of total DNA or 100 CFU/ml. The LAMP assays were applied to a total of 155 samples of direct DNA extraction from sputum and hemoculture bottles. The sensitivity of LAMP for mecA detection in sputum and hemoculture bottles was 93.3% (28/30) and 100% (52/52), respectively. In conclusion, LAMP assay is an alternative technique for rapid detection of MRSA infection with a technical simplicity and cost-effective method in a routine diagnostic laboratory.
Asunto(s)
Técnicas Bacteriológicas/métodos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Reología , Esputo/microbiología , Secuencia de Bases , Biotina/metabolismo , Cartilla de ADN/metabolismo , Electroforesis en Gel de Agar , Genes Bacterianos , Humanos , Límite de Detección , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA.
