Comparison of stabilisers for development of a lyophilised multiplex reverse-transcription PCR mixture for rapid detection of foot and mouth disease virus serotypes

Abstract

Multiplex reverse-transcription polymerase chain reaction (mRT-PCR) assay is a sensitive and rapid method for the detection and serotyping of foot and mouth disease virus (FMDV). However, the method has not been used to its full potential, because of factors such as cost, a lack of infrastructure and the complexity of the reaction mixture. This study was undertaken to optimise and validate a thermostable, lyophilised, ready-to-use mRT-PCR kit for the rapid detection of FMDV in field laboratories in India. Trehalose, PEG-8000 and glycerol were evaluated for stabilisation of the PCR mixture at ambient temperatures. The lyophilised mRT-PCR kit was validated and found robust enough for use in field-level laboratories. The PCR reaction mixture in the ready-to-use kit has low complexity, so chances of cross-contamination during the preparation of the mixture are limited, but may easily be monitored by using lyophilised internal positive and negative controls. In addition, the requirement to maintain live FMDV isolates as internal positive controls at field-level regional laboratories is eliminated. KeywordsFoot and mouth disease -India -Lyophilised RT-PCR reagent -Ready-to-use RT-PCR kit.

Optimization of Lyophilized LAMP and RT-PCR Reaction Mixes for Detection of Tuberculosis

“…Accordingly, the cryoprotectant given in table 1 was tested at the indicated concentrations. As shown in figure 2, the cryoprotectant mixture proposed by Sharma et al, (2014) and Klatser et al, (1998) (20,24) has not been successful in our studies ( Fig. 1 and Fig.…”

“…For the lyophilization of LAMP and RT-PCR mixtures, three different cryoprotectant mixtures were tested. In the studies carried out as a result of the lyophilization process, described by Sharma et al, (2014) and Klatser et al, (1998) (20,24) contents were found to cause inhibition in both amplification reactions which can be shown in figure 2. The cryoprotectant mixture tested for the first time in this study is suitable for LAMP and RT-PCR studies.…”

“…Two different cryoprotectant mixtures which described by Sharma et al (2014) and Klatser et al (1998) (20,24), except our mixture was used for LAMP and RT-PCR methods. The tested lyophilization contents in this study are given in table 1.…”

Development of Freeze-dried Reagents based Multiplex PCR Assay for the Detection of Common and Emerging Abortion-causing Pathogens

“…Virus isolates of BHV-1 and BTV10 were revived in MDBK and BHK-21 cells, respectively. With the appearance of obvious cytopathic effects (CPE), viruses were harvested and subjected to the endpoint dilution method of titration as described by Spearman and Karber 19,20 . Total nucleic acid (TNA) was extracted from virus isolates with a pre-determined titer, using magnetic beads-based MagMax TNA extraction kit (Thermo Scientific), as per the manufacturer's instructions.…”