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Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides
Citation:
Ware, M., S. Keely, AND E. Villegas. Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 115(1):298-309, (2013).
Impact/Purpose:
This off-the-slide genotyping technique is a simple and low cost tool that expands the applications of USEPA Method 1623 results by identifying the genotypes and Assemblages of the enumerated Cryptosporidium and Giardia. This additional information can be used to develop more accurate microbial risk assessment models, implement better watershed management decisions, and improve future regulations of these pathogens.
Description:
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. Transfer efficiency from the slide to the PCR tube was not possible by microscopy, but revealed that the transfer process aided in the physical lysis of the (oo)cysts releasing DNA. PCR detection rates for a single event on a slide were 44 % for Giardia and 27% for Cryptosporidium, a minimum of 5 cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR densities and limits of detection showed that 18 Cryptosporidium and 5 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 a 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 USEPA Method 1623 results by identifying the genotypes and Assemblages of the enumerated Cryptosporidium and Giardia. This additional information can be used to develop more accurate microbial risk assessment models, implement better watershed management decisions, and improve future regulations of these pathogens.
