Evaluation of Chlamydophila abortus DNA extraction protocols for polymerase chain reaction diagnosis in paraffin-embedded tissues.
Polymerase chain reaction (PCR) has gained increasing importance as a tool for directly demonstrating the presence of Chlamydophila in the placentas of aborted sheep and goats. However, because of the zoonotic potential of the disease, it is advisable to use fixed materials. To evaluate 4 different DNA extraction protocols in paraffin-embedded sections for PCR, previously immunohistochemically diagnosed placental samples from outbreaks of abortions in goats and sheep were used. The samples were also used to evaluate the effect of the duration of fixation in formalin on PCR. A protocol that uses Tris-HCl pH 8.5 with EDTA and subsequent digestion with proteinase K was found to be an easy protocol for obtaining excellent PCR products for Chlamydophila abortus diagnosis from formalin-fixed and paraffin-embedded specimens. It was also found that if samples are fixed in formalin for more than 2 weeks, the PCR technique is affected more adversely than immunohistochemical methods.
Ortega
Nieves
N
Departmento de Sanidad Animal, Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain.
Navarro
José A
JA
Nicolás
Laura
L
Buendía
Antonio J
AJ
Caro
María R
MR
Del Río
Laura
L
Martínez
Carlos M
CM
Cuello
Francisco
F
Salinas
Jesús
J
Gallego
María C
MC
D000034:Abortion, Veterinary / Q000175:diagnosis / Q000382:microbiology; D000818:Animals; D023501:Chlamydophila / Q000235:genetics*; D023521:Chlamydophila Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D015511:Goat Diseases / Q000175:diagnosis / Q000382:microbiology; D006041:Goats; D016612:Paraffin Embedding / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000382:microbiology
D004269:DNA, Bacterial