Comparison of three different preservatives for morphological and real-time PCR analyses of Haemonchus contortus eggs.
Despite the development of several recent PCR assays for the egg stages of various trichostrongyles, there have been no protocols described for preserving field samples for PCR without refrigeration. In this study, Lugol's iodine (LI), sodium azide (SA), and neutral buffered formalin (NBF) were evaluated using Haemonchus contortus eggs to determine their potential as a preservative for trichostrongyle egg samples to be processed with real-time PCR. When egg recovery, embryo development, and egg morphology were evaluated from fecal samples preserved with LI, NBF, and SA, there was equally good recovery and preservation for the first month. Preserved eggs were detectable for 1 month, but after 6 months, none could be recovered. When real-time PCR analysis was performed on eggs isolated from faeces preserved with LI and SA, there was no detectable inhibition compared to fresh, non-preserved eggs; however, NBF significantly inhibited amplification. The results from this study demonstrate that for PCR applications LI and SA are effective preservatives for H. contortus eggs, resulting in good preservation of morphology while allowing for uninhibited PCR.
Harmon
Aaron F
AF
Department of Biology & Microbiology, South Dakota State University, Brookings, SD 57007, USA.
Williams
Zachary B
ZB
Holler
Larry D
LD
Hildreth
Michael B
MB
D000818:Animals; D005404:Fixatives; D005557:Formaldehyde; D006189:Haemonchus / Q000166:cytology* / Q000235:genetics*; D007454:Iodides; D010063:Ovum / Q000166:cytology* / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011309:Preservation, Biological / Q000379:methods*; D019810:Sodium Azide
D005404:Fixatives; D007454:Iodides; D005557:Formaldehyde; D019810:Sodium Azide; C010389:Lugol's solution