Laboratory Optimization Tweaks for Sanger Sequencing in a Resource-Limited Setting.
J Biomol Tech
; 31(4): 157-164, 2020 12.
Article
en En
| MEDLINE
| ID: mdl-33100921
ABSTRACT
Despite various challenges that hinder the implementation of high-tech molecular methods in resource-limited settings, we have been able to implement and achieve International Organization for Standardization 151892012 accreditation for genotypic HIV drug resistance testing in our facility. At the Center for Human Virology and Genomics, Nigerian Institute of Medical Research, Nigeria has recorded a high sequencing success rate and good quality sequence data. This was achieved by optimizing laboratory processes from 2008 to the current date. We have optimized sample preparation, RT-PCR, several post-PCR processes, and the cycle sequencing to improve the sensitivity of amplification even with limited plasma samples and low viral copy numbers. The optimized workflow maximizes output, minimizes reagent wastage, and achieves substantial cost savings without compromising the quality of the sequence data. Our performance at our last external quality assurance program is a testimonial to the efficiency of the workflow. For the 5-sample panel, each with 67-68 mutation points evaluated, we scored 100% for all 5 specimens. Our optimized laboratory workflow is thus documented to support laboratories and to help researchers achieve excellent results the first time and eliminate contamination while minimizing the wastage of costly sequencing reagents.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
ARN Viral
/
Infecciones por VIH
/
VIH-1
/
Análisis de Secuencia de ADN
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Farmacorresistencia Viral
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Reacción en Cadena en Tiempo Real de la Polimerasa
Tipo de estudio:
Prognostic_studies
Límite:
Humans
País como asunto:
Africa
Idioma:
En
Año:
2020
Tipo del documento:
Article