Rapid non-enzymatic extraction method for isolating PCR-quality camelpox virus DNA from skin.
Molecular diagnostic investigations of orthopoxvirus (OPV) infections are performed using a variety of clinical samples including skin lesions, tissues from internal organs, blood and secretions. Skin samples are particularly convenient for rapid diagnosis and molecular epidemiological investigations of camelpox virus (CMLV). Classical extraction procedures and commercial spin-column-based kits are time consuming, relatively expensive, and require multiple extraction and purification steps in addition to proteinase K digestion. A rapid non-enzymatic procedure for extracting CMLV DNA from dried scabs or pox lesions was developed to overcome some of the limitations of the available DNA extraction techniques. The procedure requires as little as 10mg of tissue and produces highly purified DNA [OD(260)/OD(280) ratios between 1.47 and 1.79] with concentrations ranging from 6.5 to 16 microg/ml. The extracted CMLV DNA was proven suitable for virus-specific qualitative and, semi-quantitative PCR applications. Compared to spin-column and conventional viral DNA extraction techniques, the two-step extraction procedure saves money and time, and retains the potential for automation without compromising CMLV PCR sensitivity.
Yousif
A Ausama
AA
Central Biotechnology Laboratory, College of Veterinary Medicine and Animal Resources, Bldg. 999, King Faisal University, Al-Hufof, 31982, Al-Ahsaa, Saudi Arabia. ausama_yousif@yahoo.com
Al-Naeem
A Abdelmohsen
AA
Al-Ali
M Ahmad
MA
D000818:Animals; D002162:Camelus; D004279:DNA, Viral / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D018155:Orthopoxvirus / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D011213:Poxviridae Infections / Q000662:veterinary* / Q000821:virology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012867:Skin / Q000821:virology*; D014773:Virology / Q000379:methods*
D004279:DNA, Viral