RESUMEN
BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.
Asunto(s)
Betacoronavirus/genética , Infecciones por Coronavirus/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nasofaringe/virología , Neumonía Viral/diagnóstico , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Automatización , COVID-19 , Prueba de COVID-19 , Chlorocebus aethiops , Técnicas de Laboratorio Clínico , Proteínas de la Envoltura de Coronavirus , Proteínas de la Nucleocápside de Coronavirus , ARN Polimerasa Dependiente de ARN de Coronavirus , Virus de la Encefalomiocarditis/genética , Humanos , Levivirus/genética , Proteínas de la Nucleocápside/genética , Pandemias , Fosfoproteínas , ARN Viral/análisis , ARN Polimerasa Dependiente del ARN/genética , SARS-CoV-2 , Análisis de Secuencia de ARN , Glicoproteína de la Espiga del Coronavirus/genética , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
OBJECTIVE: To determine the accuracy of the current oxacillin resistant Staphylococcus aureus (S. aureus) detection test used in Makkah hospitals in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) method. METHODS: A total of 500 S. aureus clinical isolates and it's oxacillin sensitivity patterns were collected from the 4 main hospitals in Makkah, Kingdom of Saudi Arabia between April 2003 and January 2004. The oxacillin sensitivity of these clinical isolates were re-examined using the NCCLS standard method and confirmed using polymerase chain reaction (PCR) technique. RESULTS: Of 500 clinical isolates, 103 (20.6%) were resistant to oxacillin using NCCLS standard method but they were sensitive according to the current hospital routine sensitivity test method. The PCR technique confirmed the presence of mecA gene in 88/103 isolates appeared to be methicillin-resistant S. aureus (MRSA) using NCCLS standard technique. CONCLUSION: A significant percentage of MRSA are currently misdiagnosed in accordance with the current routine sensitivity method. In addition, some mecA negative and oxacillin resistant strains (according to the NCCLS standard method) can be misdiagnosed by using PCR technique. These findings emphasis the urgent need to comply with the recommended NCCLS guidelines for detection of oxacillin resistance. Moreover, the PCR technique can not be used as a single diagnostic tool for detection of MRSA.
