PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

Coxiella PCR 155
3.2.1. Vaginal Swabs
Vaginal secretions, sampled immediately after abortion by swabbing provide
a suitable material for isolation and PCR analysis of abortive organisms.
They are not usually as heavily infected as the cotyledons, but they reflect
moderate infection of the placenta and are less hazardous to the handler.
Samples should be collected as soon as possible after abortion. Vaginal excretion,
often abundant during the first few days, can decrease rapidly or become
intermittent making testing inaccurate. Vaginal swabs are taken by the insertion
of a dry, sterile cotton wool swab (10 cm) into the vagina of each animal
and should be sent to the laboratory as they are without any transport medium.
3.2.2. Milk
Colostrum and milk from the quarters should be collected aseptically in a
sterile container. Before sampling, the teats have to be cleaned, and the first
two jets of milk should be discarded. The milk sample may stored at 20°C
before use.
3.2.3. Feces
As for milk samples, fecal material should also be taken directly from the
animal and collected in a sterile container. If the samples are not examined
immediately, they must be stored at 20°C.
3.2.4. Placental Tissue
The placenta, when available and not soiled, is the best sample for detection
of the abortive agents (see Note 1). Since the entire placenta is difficult and
hazardous to handle, it is better to sample the cotyledons. Thus, 5 or 6 cotyledons
and their intercotyledonary membranes should be collected and placed in
a sterile watertight container. Rinse them with physiological saline and dry
with filter paper. The samples may be stored at 4°C for several days or at
20°C for several months.
3.3. DNA Extraction from Different Types of Samples
The sample preparation steps are performed in a confined level L3 laboratory.
The DNA extraction steps can be conducted outside the safety laboratory
after proteinase K treatment.
3.3.1. DNA Extraction from C. burnetii Nine-Mile Reference Strain
1. Boil 100 µL of the bacterial suspension for 10 min.
2. Centrifuge the solution at 13,000g for 5 min.
3. After centrifugation, the supernatant can be used either as a positive amplification
control or for the evaluation of the sensitivity of the PCR assay.