A duplex, SYBR Green I-based RT-qPCR assay for the simultaneous detection of Apple chlorotic leaf spot virus and Cherry green ring mottle virus in peach.
BACKGROUND
Co-infections of Apple chlorotic leaf spot virus (ACLSV) and Cherry green ring mottle virus (CGRMV) in peach is common in China and have resulted in significant yield reductions. A reliable, sensitive and quantitive method is needed to detect and distinguish between ACLSV and CGRMV in peach.


RESULTS
We developed a sensitive and specific SYBR Green-I based RT-qPCR for the quantification of ACLSV and CGRMV in different peach tissues, and a duplex RT-qPCR system to detect ACLSV and CGRMV simultaneously. The RT-qPCR method was optimized using standard samples transcribed by the T7 Large Scale RNA Production System in vitro. The peach genes, RNA Polymerase subunit II (RPII) and Ubiquitin 10 (UBQ10), which were used as the internal controls for the quantification assay also showed good expression stability in this system. Single RT-qPCR assays showed that CGRMV in peach accumulates to a higher level than ACLSV. The detection limits of the duplex RT-qPCR assay were 10² and 10⁴ copies for ACLSV and CGRMV, respectively. The sensitivity of the duplex RT-qPCR was as high as RT-qPCR and higher than RT-PCR.


CONCLUSIONS
The SYBR Green-I RT-qPCR assay provided a sensitive, specific and reliable method for the detection and quantification of ACLSV and CGRMV in different peach tissues. The duplex RT-qPCR system provided a sensitive and specific method to detect and differentiate between ACLSV and CGRMV in a single sample. This RT-qPCR assay could be a useful tool for the routine diagnosis of these two viruses and for disease epidemiology studies in peach orchards.
College of Agronomy and Biotechnology, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing 100193, People's Republic of China.
Zhe
Z
Zhao
Yun
Y
Yu
Zhixiang
Z
Zhang
Pengbo
P
Liang
Yuxin
Y
Ma
Shifang
S
Li
Hongqing
H
Wang
D052160:Benzothiazoles; D002681:China; D017790:Closterovirus / Q000235:genetics / Q000302:isolation & purification*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003959:Diamines; D052677:Flexiviridae / Q000235:genetics / Q000302:isolation & purification*; D009930:Organic Chemicals / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology*; D027861:Prunus / Q000821:virology*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D025801:Ubiquitin / Q000235:genetics; D019562:Viral Load / Q000379:methods* / Q000592:standards
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D025801:Ubiquitin; C098022:SYBR Green I; D012321:DNA-Directed RNA Polymerases