M W Ware1, S P Keely, E N Villegas. 1. National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, OH 45268, USA.
Abstract
AIMS: This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. METHODS AND RESULTS: Slide standards containing flow-sorted (oo)cysts were used to evaluate the off-the-slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide. CONCLUSIONS: This study successfully developed and evaluated recovery rates and limits of detection of an off-the-slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide. SIGNIFICANCE AND IMPACT OF THE STUDY: This off-the-slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions. Journal of Applied Microbiology Published [2013]. This article is a U.S. Government work and is in the public domain in the USA.
AIMS: This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. METHODS AND RESULTS: Slide standards containing flow-sorted (oo)cysts were used to evaluate the off-the-slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide. CONCLUSIONS: This study successfully developed and evaluated recovery rates and limits of detection of an off-the-slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide. SIGNIFICANCE AND IMPACT OF THE STUDY: This off-the-slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions. Journal of Applied Microbiology Published [2013]. This article is a U.S. Government work and is in the public domain in the USA.
Authors: Hodon Ryu; Yao Addor; Nichole E Brinkman; Michael W Ware; Laura Boczek; Jill Hoelle; Jatin H Mistry; Scott P Keely; Eric N Villegas Journal: Water (Basel) Date: 2021-05-21 Impact factor: 3.103