不同DNA提取法对腺病毒和细小病毒PCR检测效果评估

EVALUATION OF SENSTITIVITY OF PCR AMPLIFICATION FOR ADENOVIRUS AND PORCINE PARVOVIURS DETECTION BY THREE DIFFERENT METHODS

分别利用硫氰酸胍(Guanidine thiocyanate,GuScN)抽提法、螯合树脂处理法和蛋白酶K-酚/氯仿抽提法从粪便样品中制备腺病毒DNA(dsDNA)或细小病毒DNA(ssDNA)然后进行PCR检测。结果显示硫氰酸胍抽提法、螯合树脂处理法能有效地去除粪便中影响PCR扩增的抑制物,提高PCR检测的敏感性,而传统蛋白酶K-酚/氯仿抽提法不能有效地去除PCR扩增的抑制物,影响PCR检测结果。在检测粪便中腺病毒时,硫氰酸胍、螯合树脂和蛋白酶-酚/氯仿抽提法分别允许检测5TCID50、50TCID50、50TCID50的病毒滴度。在用硫氰酸胍、螯合树脂和蛋白酶-酚/氯仿抽提法制备粪便中猪细小病毒核酸进行PCR检测时,3种方法分别可检测10TCID50、50TCID50、100TCID50的病毒滴度。结果也显示硫氰酸胍螯合树脂制备法具有价格便宜、检测速度快、灵敏度高等特点,适合于临床检测。

Using specimens spiked with adenovirus or porcine parvovirus, several DNA extraction methods were evaluated for their ability to remove polymerase chain reaction (PCR) inhibitors from stool samples. It was found that PCR inhibition could be partially overcome by extracting viral DNA with guanidine thiocyanate ( GuScN) or chelex resin methods, but PCR inhibition could be hardly overcome by extracting viral DNA with traditional proteinase-K phenol chloroform extraction method. For adenoviurs (dsDNA) in stool samples, GuScN, chelex resin, proteinase-K phenol chloroform methods respectively allowed 5 TCID50, 50TCID50, 50 TCID50 titer to be detected. For porcine parvovirus (ssD-NA) in stool samples, three protocols respectively allowed 10 TCID50, 50 TCD50, 100 TCID50 to be detected. . Their rapidity and low cost make GuScN and chelex resin extraction methods the suitable for routine diagnostic testing.