液体培养法与qPCR法对检测猪鼻支原体灵敏度比较

Comparison of Detection Sensitivity for Mycoplasma Hyorhinis Between Liquid Culture and qPCR

为了比较《中国药典》已收录但相当耗时的液体培养法和未收录但相对快速的荧光定量PCR(qPCR)法对支原体检测的灵敏度,试验分别在猪鼻支原体生长的对数期(48 h)、稳定期(96 h)和衰亡期(144 h)取样,将样品10倍系列稀释后,使用液体培养法和qPCR法对各个稀释度进行检测(两种方法的样品加入体积分别为0.5 mL和5μL),以比较二者的灵敏度。结果显示:在存活支原体占比高的对数期和稳定期,液体培养法比qPCR法的检测灵敏度高约100倍;而在死亡支原体占比大幅增加的衰亡期,结果则相反,qPCR法反而比液体培养法的检测灵敏度高约1 000倍。当把对数期样品高速离心浓缩约100倍后再用qPCR法检测,其检测灵敏度比浓缩之前提高了约100倍。上述结果表明:影响液体培养法和qPCR法相对灵敏度的主要因素是各自体系中不同的样品加入体积和样品中存活支原体的比例,而由于前一因素导致的两种方法灵敏度的差异,可以通过高速离心浓缩的方法缩小,使两种方法的相对灵敏度基本相同。

To Compare the detection sensitivity between liquid culture and real􀆼time qPCR for the samples collected at different growth phases of Mycoplasma hyorhinis(included in the Chinese Pharmacopoeia but very time􀆼consuming)(not included in the Chinese Pharmacopoeia but rapid),samples were collected at three different growth phases of Mycoplasma hyorhinis:log phase(48 h),stationary phase(96 h)and dead phase(144 h).Then the samples were diluted 10 times serially,and the detection sensitivity was tested by liquid culture and qPCR.The results showed that the detection sensitivity of liquid culture was about 100 times higher than that of the qPCR method in the log phase and stationary phase with a high proportion of viable mycoplasma.In the dead phase when the proportion of dead mycoplasma increased significantly,the detection sensitivity of qPCR was about 1000 times higher than that of liquid culture.When the log phase sample was concentrated(about 100 times)by centrifugation,they were detected again by qPCR.Compared with the sample before concentration,the detection sensitivity of the sample was increased by 100 times.In conclusion,the main factors affecting the relative detection sensitivity of liquid culture and qPCR are the sample addition volumes in their respective systems and the proportion of viable and dead mycoplasma cells in the samples.The difference in sensitivity between the two methods caused by the former factor can be reduced by high􀆼speed centrifugal concentration,so that the sensitivity of the two methods is the same.