Microsporidia can cause serious disease and are common environmental organisms that have
been implicated in waterborne disease outbreaks so attempts have been made to recover
microsporidia from water samples for subsequent testing with traditional PCR. Unfortunately,
existing molecular procedures are largely uncharacterized, labor intensive, and do not lend
themselves to routine use in a water utility laboratory. These experiments addressed these
limitations by using an automated nucleic acid extraction method, the MagNAPure LC
workstation (MPLC), and a rapid real-time PCR assay, the Encephalitozoon LC assay (LC -
PCR) for the LightCycler(TM) instrument (Roche Molecular Biochemicals, Penzburg, Germany).
Using laboratory water spiked with spores, a modification of the MagNA Pure LC DNA Isolation
Kit I protocol was performed using the automated MPLC. Extracted DNA was amplified using
the LC-PCR. Assay detection limits, reproducibility, and efficiency were assessed by comparing
threshold cross-over values generated by the LC-PCR. The assay provided rapid reproducible
results for detection of microsporidia in laboratory water. The lower limit of detection (LLOD) for
spores was less than or equal to 100 spores/ml of laboratory water (i.e., 0.5 spores/ml). PCR
efficiency approached 100% for spores in water and was statistically equivalent for spore
dilutions 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-assay
reproducibility was assessed at 104 spores/ml of water and produced a mean C.O. of 33.2 +/- 0.9
with a CV of 2.7 (n=8). Stability of the control spores at 4C was assessed by monitoring the
C.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). By
using 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 C
respectively (n=3). Except for E. intestinalis strains, all Tm were significantly different as judged
by the 95% CI. No differences were observed in Tm as a function of spore concentration. While
there are several published molecular detection assays for microsporidia species in water, this
real-time PCR is used to characterize analytical accuracy, reproducibility, and efficiency as well
as to enable the differentiation of Encephalitozoon species in a single homogenous real-time
PCR system. These methods have the potential to be adapted and safely incorporated into a
reference laboratory and provide the water industry with a standardized approach by which
other methods can be compared. This method can provide the basis for further testing of
different water matrices and concentrated water pellets for the detection of microsporidia in
source water.
Includes 42 references.
| Edition : | Vol. - No. |
| File Size : | 1
file
, 320 KB |
| Note : | This product is unavailable in Ukraine, Russia, Belarus |
| Number of Pages : | 9 |
| Published : | 11/01/2002 |
