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

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13. Lysis buffer: 100 mM Tris-HCl, 0.05 % Tween 20, 0.2 mg/mL proteinase K, pH 8.5.
14. dATP 100 mM, dCTP 100 mM, dGTP 100 mM, dTTP 100 mM.
15. Taq DNA polymerase 5 U/µl.
3. Methods
The methods described below are aimed at (i) the identification and toxin gene
typing of A. pleuropneumoniae isolates from cultures and (ii) the direct detection
of A. pleuropneumoniae by PCR in lung tissue and nasal secretory fluid.
3.1. Identification and Toxin Gene Typing
Identification of the species A. pleuropneumoniae and toxin gene typing of
an isolate or a strain from culture on solid medium (e.g., blood agar, chocolate
agar, or Columbia agar-βNAD) is performed by preparing a lysate of the culture
followed by six individual PCRs using the lysed bacteria as template. As
positive controls, lysates from A. pleuropneumoniae cultures of serotype 1
strain 4074 and serotype 2 strain S1536 are used.
3.1.1. Preparation of Lysates
Lysates are made from all strains to be tested and from each of the two
control strains.
1. Add 450 µL lysis buffer to an Eppendorf tube.
2. Resuspend 4 to 5 bacterial colonies.
3. Vortex mix strongly, to obtain a good suspension.
4. Incubate tube at 60°C for 60 min (heating block).
5. Vortex mix strongly.
6. Incubate at 95°–97°C for 15 min (inactivate proteinase).
7. Use lysates directly as templates for PCR, or store at 20°C.
3.1.2. Preparation of PCR Premix
For each specific PCR (see Table 2), 1 mL PCR premixture is first prepared
in the safety cabinet, taking all necessary precautions to prevent contamination.
Then, PCRs are made for each of the genes apxICA, apxIICA, apxIIICA,
apxIBD, apxIIIBD, and for apxIVA (see Note 1). The PCR based on apxIV is
made to confirm the species A. pleuropneumoniae (see Notes 2 and 3).
PCR Premixture (label tube with corresponding gene pairs to be amplified):
H 2O bidistilled 843 µL
10X PCR buffer 100 µL
dATP 100 mM 1.7 µL
dCTP 100 mM 1.7 µL
dGTP 100 mM 1.7 µL
dTTP 100 mM 1.7 µL
Primer-L 10 mM (see Table 2) 25 µL
Primer-R 10 mM(see Table 2) 25 µL