Schwartz I, Varde S, Nadelman R B, Wormser G P, Fish D
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, USA.
Am J Trop Med Hyg. 1997 Mar;56(3):339-42. doi: 10.4269/ajtmh.1997.56.339.
Polymerase chain reaction (PCR) analysis is a widely used method for detection of Borrelia burgdorferi DNA in biological specimens, including ticks. Studies have demonstrated that substances present in mammalian blood can inhibit PCR amplification. This would limit the utility of PCR for determination of B. burgdorferi infection in engorged ticks that have taken a blood meal from a human or other animal host. To systematically assess the potential for such inhibition, nymphal Ixodes scapularis, which had acquired B. burgdorferi as larvae, were fed on rats. These engorged ticks were lysed in standard PCR lysis buffer and aliquots were subjected to PCR analysis; 0 of 56 were PCR positive. An equivalent cohort of unfed (unengorged) ticks had an infection rate of 19% (11 of 57) as determined by identical PCR analysis (P = 0.0006, by Fisher's exact test). When lysates from the engorged ticks were spiked with the 500 prelysed B. burgdorferi, none of the samples yielded a positive PCR signal, indicating the presence of inhibitory substances. Consistent with this observation, PCR amplification of the original engorged tick lysates after extraction with a DNA extraction kit, resulted in detection of B. burgdorferi DNA in 13 specimens (23%). Furthermore, when 500 prelysed B. burgdorferi were added to the treated extracts, all samples (56 of 56) were PCR positive. Thus, extraction resulted in removal of inhibitors of PCR amplification present in unprocessed engorged tick lysates. Furthermore, additional titration experiments showed that some inhibitory substances may also be present in unfed ticks, although this inhibition does not completely prevent detection of B. burgdorferi DNA in unprocessed lysates. This study clearly demonstrates that inhibitors of PCR amplification are present in engorged ticks and prevent accurate determination of B. burgdorferi infection rates by this method unless steps are taken to remove such inhibitors.
聚合酶链反应（PCR）分析是一种广泛用于检测生物标本（包括蜱虫）中伯氏疏螺旋体DNA的方法。研究表明，哺乳动物血液中存在的物质会抑制PCR扩增。这将限制PCR在确定已从人类或其他动物宿主吸血的饱血蜱中伯氏疏螺旋体感染情况的应用。为了系统评估这种抑制的可能性，将幼虫期已感染伯氏疏螺旋体的肩突硬蜱若虫喂以大鼠。这些饱血蜱在标准PCR裂解缓冲液中裂解，取等分试样进行PCR分析；56只中0只为PCR阳性。通过相同的PCR分析，一组同等数量的未进食（未饱血）蜱的感染率为19%（57只中有11只）（Fisher精确检验，P = 0.0006）。当向饱血蜱的裂解物中加入500个预先裂解的伯氏疏螺旋体时，没有一个样品产生阳性PCR信号，表明存在抑制物质。与该观察结果一致，用DNA提取试剂盒提取原始饱血蜱裂解物后进行PCR扩增，在13个标本（23%）中检测到了伯氏疏螺旋体DNA。此外，当向处理后的提取物中加入500个预先裂解的伯氏疏螺旋体时，所有样品（56只中的56只）均为PCR阳性。因此，提取去除了未处理的饱血蜱裂解物中存在的PCR扩增抑制剂。此外，额外的滴定实验表明，未进食的蜱中也可能存在一些抑制物质，尽管这种抑制并未完全阻止在未处理的裂解物中检测到伯氏疏螺旋体DNA。这项研究清楚地表明，饱血蜱中存在PCR扩增抑制剂，除非采取措施去除这些抑制剂，否则会妨碍用该方法准确测定伯氏疏螺旋体感染率。
