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DOI10.1111/jam.12223
Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides
Ware, M. W.; Keely, S. P.; Villegas, E. N.
发表日期2013-07-01
ISSN1364-5072
卷号115期号:1页码:298-309
英文摘要

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.


英文关键词Cryptosporidium;cysts;genotyping;Giardia;oocysts;US EPA Method 1623
语种英语
WOS记录号WOS:000320278600031
来源期刊JOURNAL OF APPLIED MICROBIOLOGY
来源机构美国环保署
文献类型期刊论文
条目标识符http://gcip.llas.ac.cn/handle/2XKMVOVA/56617
作者单位US EPA, Natl Exposure Res Lab, Cincinnati, OH 45268 USA
推荐引用方式
GB/T 7714
Ware, M. W.,Keely, S. P.,Villegas, E. N.. Development and evaluation of an off-the-slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides[J]. 美国环保署,2013,115(1):298-309.
APA Ware, M. W.,Keely, S. P.,&Villegas, E. N..(2013).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,115(1),298-309.
MLA Ware, M. W.,et al."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 115.1(2013):298-309.
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