Detection of enteroviruses and hepatitis A virus in water by consensus primer multiplex RT-PCR.
OBJECTIVE
To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV).


METHODS
A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-,671-, 1084-, and 1128bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV.


RESULTS
All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus,21 PFU for Coxsackie virus,60 PFU for Echovirus and 105 TCID(50) for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID(50) for HAV.


CONCLUSIONS
The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness.
Li
Jun-Wen
JW
Department of Environment and Health, Institute of Health and Environmental Medicine of Tjianjin, 1 Da Li Road, Tianjin 300050, China. junwenli@eyou.com
Wang
Xin-Wei
XW
Yuan
Chang-Qing
CQ
Zheng
Jin-Lai
JL
Jin
Min
M
Song
Nong
N
Shi
Xiu-Quan
XQ
Chao
Fu-Huan
FH
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D029822:Enterovirus B, Human / Q000235:genetics / Q000302:isolation & purification; D030041:Hepatitis A virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D017931:DNA Primers; D012367:RNA, Viral