Simple template-based reverse transcription-recombinase polymerase amplification assay for routine diagnosis of citrus tristeza virus.

This study reports a simple template-based reverse transcription-polymerase amplification assay (ST-RT-RPA) for detection of citrus tristeza virus (CTV) from crude plant extract lysed in NaOH:EDTA (1:1) without the need of tedious RNA isolation. The developed assay showed versatility in its usage as amplification can be performed at wide temperature range (14°C to 42°C) and incubation time (4 to 32 min), although the best conditions were 38°C for 30 min. The developed ST-RT-RPA assay could detect the CTV up to 10-8 dilution of crude plant extract of NaOH:EDTA and up to 0.01 fg µl-1 of RNA of CTV-infected plant tissues and 0.001 ag µl-1 of plasmid DNA containing viral insert, thus exhibiting sufficient sensitivity. ST-RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus yellow vein clearing virus, and Candidatus Liberibacter asiaticus) and was more sensitive in detection of CTV infection in field samples as compared to standard reverse transcription-polymerase chain reaction (RT-PCR) with later showing false negative in 7.92% of samples tested after 1 week of sampling. The developed ST-RT-RPA assay used minimally processed crude plant extract as template, tolerant to sample degradation in transit and storage, while it can be easily performed at wide temperatures and could be adopted in resource-poor setup.

Prevalence, toxinotyping, antimicrobial susceptibility and biofilm-forming ability of Clostridium perfringens isolated from free-living rodents and shrews.

BACKGROUND AND OBJECTIVES: Clostridium perfringens (C. perfringens), is a spore-forming and toxin-producing pathogenic Gram-positive rod-shaped bacterium with immense public health/zoonotic concern. Rodents are well-known reservoirs and vectors for a large number of zoonoses and strong links have been recognized between synanthropic rodents and foodborne disease outbreaks throughout the world. To date, no study has been conducted for studying the prevalence of C. perfringens in rodents and shrews. In this study, we investigated faecal samples from free-living rodents and shrews trapped in Meghalaya, a North-eastern hill state of India for the presence of virulent and antimicrobial-resistant C. perfringens. METHODS: A total of 122 animals comprising six species of rodents and one species of shrews were trapped: Mus musculus (n = 15), Mus booduga (n = 7), Rattus rattus (n = 9), Rattus norvegicus (n = 3), Bandicota indica (n = 30), Bandicota bengalensis (n = 32) and Suncus murinus (n = 26). The faecal swabs were collected and processed for the isolation of C. perfringens. Toxinotyping was done using PCR. Antimicrobial susceptibility testing and biofilm forming ability testing were done using Kirby Bauer disc diffusion method and crystal violet assay. RESULTS: C. perfringens was isolated from 27 of the 122 faecal swabs (22.1%), from six species of rodents and shrews. Five of the host species were rodents, Bandicota bengalensis (25%), Bandicota indica (16.7%), Rattus norvegicus (33.3%), Mus musculus (13.3%), Mus booduga (42.8%) and Suncus murinus (shrew) (29.6%). The common toxinotype was type A (59.2%) followed by Type A with beta2 toxin (33.3%), Type C (3.7%) and Type C with beta2 toxin (3.7%). None of the isolates harboured cpe, etx, iap, and NetB genes and therefore none was typed as either B, D, E, F, or G. Nine isolates (33.3%) turned out to be multi-drug resistant (MDR), displaying resistance to three or more categories of antibiotics tested. Twenty-three out of twenty-seven isolates (85.2%) were forming biofilms. CONCLUSION: Globally, this is the first study to report the prevalence of C. perfringens and its virulence profile and antimicrobial resistance in free-living rodents and shrews. The rodents and shrews can potentially contaminate the food and environment and can infect humans and livestock with multi-drug resistant/virulent Type A and Type C C. perfringens.