A simple preparation of mycoplasmal DNA template for PCR from biological samples using effective surfactants.
To prepare mycoplasmal DNA template for PCR from biological samples rapidly and easily, surfactants which can solubilize cell membranes effectively were investigated. 3-[3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was considered an effective surfactant. This surfactant could solubilize mycoplasma cell membrane without suppressing the polymerase reaction. In addition, proteinase K treatment played an important role in preparing mycoplasmal DNA template from a simulated biological sample. It was therefore considered that a combination of proteinase K- and CHAPS- added lysis buffer would be more useful in preparing mycoplasmal DNA template. We could detect PCR products by using the lysis buffer with a simulated lung emulsion sample containing mycoplasma organisms at 10(4) CFU per gram.
Kobayashi
H
H
National Institute of Animal Health, Ibaraki, Japan.
Munthali
G
G
Miyamoto
C
C
Morozumi
T
T
Mitani
K
K
Ito
N
N
Shiono
H
H
Yamamoto
K
K
D000818:Animals; D002793:Cholic Acids; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification*; D005227:Fatty Acids; D005944:Glucosamine / Q000031:analogs & derivatives; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification*; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D013501:Surface-Active Agents; D013552:Swine; D013553:Swine Diseases; D013698:Templates, Genetic
D002793:Cholic Acids; D017931:DNA Primers; D004269:DNA, Bacterial; D005227:Fatty Acids; D013501:Surface-Active Agents; C048531:chapso; C042221:decanoyl-N-methylglucamide; D005944:Glucosamine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate