[Detection of TTV DNA from high background nuclear acid samples using specific nuclear acid captured-polymerase chain reaction].
OBJECTIVE
To improve the amplification of non-specificity of TTV DNA detection using PCR from high background nuclear acids because of its low level in samples.


METHODS
Specific nuclear acid captured PCR(SNAC-PCR) was established through capturing TTV DNA onto microplate with specific nuclear acid by means of avidin-biotin system, then amplifying captured TTV DNA as usual. Its specificity was evaluated by comparing with the detection of routine extraction samples using nested-PCR with or without subsequent DNA sequencing in 10 samples of sera and liver tissues in pairs.


RESULTS
Positive results were obtained using routine protocol in 4 and 9 out of 10 samples of the sera and the liver tissues respectively. The PCR products were proved to be specific using RFLP analysis with Kpn I. However, positive results were found in only 2/10 and 3/10 of samples of sera and liver tissue using DNA sequencing. The results of SNAC-PCR were the same as those of routine protocol with subsequent DNA sequencing. Furthermore, sharper electrophoresis bands were obtained.


CONCLUSIONS
SNAC-PCR could dramatically increase the specificity of the detection of TTV DNA, especially in high background nuclear acid samples. It can be widely used in the detection of other pathogens with low seral level or in high background nuclear acid samples. The level of TTV infection might be over-evaluated by currently used TTV DNA detection when the PCR products were not confirmed by DNA sequencing.
Peng
X
X
Department of Infectious Diseases, Third Affiliated Hospital, Sun Yat-sen University of Medical Sciences, Guangzhou 510630, China.
Deng
L
L
Chen
W
W
D001483:Base Sequence; D004266:DNA Virus Infections / Q000821:virology*; D004279:DNA, Viral / Q000097:blood*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D022783:Torque teno virus / Q000235:genetics / Q000302:isolation & purification*
D004279:DNA, Viral