Use of the LightCycler to Evaluate Extraction Methods for Detection of Microsporidia (Encephalitozoon spp.)

摘要

Microsporidia can cause serious disease and are common environmental organisms that havebeen implicated in waterborne disease outbreaks so attempts have been made to recovermicrosporidia from water samples for subsequent testing with traditional PCR. Unfortunately,existing molecular procedures are largely uncharacterized, labor intensive, and do not lendthemselves to routine use in a water utility laboratory. These experiments addressed theselimitations by using an automated nucleic acid extraction method, the MagNAPure LCworkstation (MPLC), and a rapid real-time PCR assay, the Encephalitozoon LC assay (LC -PCR) for the LightCycler? instrument (Roche Molecular Biochemicals, Penzburg, Germany).Using laboratory water spiked with spores, a modification of the MagNA Pure LC DNA IsolationKit I protocol was performed using the automated MPLC. Extracted DNA was amplified usingthe LC-PCR. Assay detection limits, reproducibility, and efficiency were assessed by comparingthreshold cross-over values generated by the LC-PCR. The assay provided rapid reproducibleresults for detection of microsporidia in laboratory water. The lower limit of detection (LLOD) forspores was less than or equal to 100 spores/ml of laboratory water (i.e., 0.5 spores/ml). PCRefficiency approached 100% for spores in water and was statistically equivalent for sporedilutions ranging from 102 - 107 spores/ml (Tukey-Kramer HSD, Dunnett's test of the means).Assay specificity was 100%, when 39 different enteric organisms were tested. Intra-assayreproducibility was assessed at 104 spores/ml of water and produced a mean C.O. of 33.2 ± 0.9with a CV of 2.7 (n=8). Stability of the control spores at 4°C was assessed by monitoring theC.O. value as a function of storage time, and proved that DNA concentration for spores in water(106 spores/ml) was relatively stable over a five-month storage period (25.4 ± 1.4 cycles). Byusing amplicon melting curve analysis, the assay reproducibly differentiated between E. Intestinalis, E. cuniculi, and E. hellem. The Tm for E. hellem, E. cuniculi, and E. intestinalis (alveolar and duodenal strains) were 63.7 ± 0.2, 67.9 ± 0.4, 69.7 ± 0.8, and 69.7 ± 0.7°Crespectively (n=3). Except for E. intestinalis strains, all Tm were significantly different as judgedby the 95% CI. No differences were observed in Tm as a function of spore concentration. Whilethere are several published molecular detection assays for microsporidia species in water, thisreal-time PCR is used to characterize analytical accuracy, reproducibility, and efficiency as wellas to enable the differentiation of Encephalitozoon species in a single homogenous real-timePCR system. These methods have the potential to be adapted and safely incorporated into areference laboratory and provide the water industry with a standardized approach by whichother methods can be compared. This method can provide the basis for further testing ofdifferent water matrices and concentrated water pellets for the detection of microsporidia insource water.