Assessing The Effects Of Different Processing Procedures On The Yield Of Treponema Pallidum DNA From Blood

Object:Polymerase chain reaction(PCR)is a method to detect the Treponema pallidum subsp.pallidum(T.pallidum)and provides definitive diagnosis of syphilis.However,the sensitivity of T.pallidum blood-PCR is moderate,giving a low level of concordance with the physician’s diagnosis.In this study,we explore a study on the effects of different clinical sample processing procedures and different sample types on the yield of T.pallidum DNA from blood in order to increase the yield of T.pallidum DNA and the positive rate of T.pallidum blood-PCR.Methods:We employed a real-time PCR with the target of Tp47 gene to evaluate the yield of T.pallidum DNA in a series of in vitro simulated experiments and the infected rabbit blood experiments with different sample processing procedures.(1)We blended a mixture system by mixing 0.1 mL of 107 treponemes/mL with anti-coagulated or non-anti-coagulated blood,and assessed the effect of red cell lysis pretreatment on the yield of T.pallidum DNA in blood samples.(2)We blended a mixture system by mixing 0.1 mL of 107 treponemes/mL with anti-coagulated blood and assessed the effect of samples processing time and storage temperature on the yield of T.pallidum DNA in blood samples.(3)We blended a mixture system by mixing 0.1 mL of 107 treponemes/mL with normal saline,serum or plasma,respectively,then extracted the DNA of different layers of the mixture system after centrifugation and assessed the effect of centrifuge force on the distribution of T.pallidum in them.(4)We blended a mixture system by mixing 0.1mL of 107 treponemes/mL with anti-coagulated or non-anti-coagulated blood,then extracted the DNA of different layers of the mixture system after centrifugation and assessed the effect of centrifuge force on the distribution of T.pallidum in them.(5)In addition,the effects of processing procedures on the yield of T.pallidum DNA was conducted in the infected rabbit blood.Results:The limit of the real-time PCR detection was approximately 3.41 copies/μL of Tp47 DNA.(1)The yield of T.pallidum DNA with red cell lysis pretreatment was 40.4 times in the whole blood,and 32.4 times in the residual haematocyte as that without pretreatment.(2)The sample processing time(within 48h)and storage temperature(between 4℃ and 25 ℃)did not have impact on the T.pallidum DNA extraction efficiency.(3)T.pallidum DNA decreased in the upper layer while increased in the lower layer with the increasing centrifuge force whether the in vitro simulated experiments or infected rabbit blood experiment.(4)For the whole blood simulated experiments in vitro,the yields of T.pallidum DNA in the lower layer were 2.8,4.6,7.3,12.6,15.24,16.7,65.1 and 73.1 times as that of the upper layer at the centrifugal force of 500×g,1000×g,2000×g,4000×g,5000×g,7000×g,10000×g and 20000×g,respectively.However,the yields of T.pallidum DNA in clot were only 1%at different centrifuge forces.(5)In addition,the infected rabbit blood experiment had a similar result when comparing with those above mentioned.Conclusions:The results show that the distribution of T.pallidum in different medium is vulnerable to the influence of centrifugal force.The yield of T.pallidum DNA can be significantly improved by appropriate centrifugation and red cell lysis pretreatment.Furthermore,the DNA extraction of T.pallidum would yield more by using whole blood or residual haematocyte when comparing with plasma,serum or clot.