RESUMO
In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Bactérias/genética , Bactérias/isolamento & purificação , Centers for Disease Control and Prevention, U.S. , Humanos , Microfluídica/métodos , Microfluídica/normas , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reprodutibilidade dos Testes , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Estados Unidos , Vírus/genética , Vírus/isolamento & purificaçãoRESUMO
BACKGROUND: The Molecular Virology Proficiency Testing Program at the Wadsworth Center began the assembly and distribution of influenza virus panels to US public health labs (PHLs) in 2008. The program was created to assist PHLs in assessing their performance and in meeting CLIA regulations for mandated proficiency testing (PT). OBJECTIVES: To design and distribute proficiency testing panels containing influenza A virus subtypes H1N1 and H3N2, and influenza B; when H1N1pdm09 emerged it also was incorporated into the panels. A secondary objective was to determine the best matrix for long term storage of the molecular PT samples. STUDY DESIGN: Viruses were quantitated using TCID(50) and quantitative real-time RT-PCR. Reference laboratories were enlisted to verify viral identity in the panels and to help determine viral titers to be used in the PT panels sent to PHLs. RESULTS AND CONCLUSIONS: Of the 29 laboratories that participated the first year, 27 were able to correctly identify all of the virus types in the panel. Fifty-one PHLs participated in the program the second year when pandemic H1N1 was added, and 45 were able to correctly detect, type and subtype all of the viruses in the panel. In the program's third year, 60 laboratories participated; 58 correctly detected and subtyped all of the viruses in the panel. Annual surveys of assay techniques showed that the PHLs had shifted their extraction methods and PCR-thermocycler instrumentation to meet FDA-approved methods. The degradation study revealed that frozen viral stocks were stable for at least 30months, thus allowing ample time to prepare and pre-test panels.