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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. 2013

Zhe Zhao, and Yun Yu, and Zhixiang Zhang, and Pengbo Liang, and Yuxin Ma, and Shifang Li, and Hongqing Wang
College of Agronomy and Biotechnology, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing 100193, People's Republic of China.

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.

UI MeSH Term Description Entries
D009930 Organic Chemicals A broad class of substances containing carbon and its derivatives. Many of these chemicals will frequently contain hydrogen with or without oxygen, nitrogen, sulfur, phosphorus, and other elements. They exist in either carbon chain or carbon ring form. Organic Chemical,Chemical, Organic,Chemicals, Organic
D010935 Plant Diseases Diseases of plants. Disease, Plant,Diseases, Plant,Plant Disease
D011804 Quinolines
D012015 Reference Standards A basis of value established for the measure of quantity, weight, extent or quality, e.g. weight standards, standard solutions, methods, techniques, and procedures used in diagnosis and therapy. Standard Preparations,Standards, Reference,Preparations, Standard,Standardization,Standards,Preparation, Standard,Reference Standard,Standard Preparation,Standard, Reference
D002681 China A country spanning from central Asia to the Pacific Ocean. Inner Mongolia,Manchuria,People's Republic of China,Sinkiang,Mainland China
D003959 Diamines Organic chemicals which have two amino groups in an aliphatic chain. Diamine
D012321 DNA-Directed RNA Polymerases Enzymes that catalyze DNA template-directed extension of the 3'-end of an RNA strand one nucleotide at a time. They can initiate a chain de novo. In eukaryotes, three forms of the enzyme have been distinguished on the basis of sensitivity to alpha-amanitin, and the type of RNA synthesized. (From Enzyme Nomenclature, 1992). DNA-Dependent RNA Polymerases,RNA Polymerases,Transcriptases,DNA-Directed RNA Polymerase,RNA Polymerase,Transcriptase,DNA Dependent RNA Polymerases,DNA Directed RNA Polymerase,DNA Directed RNA Polymerases,Polymerase, DNA-Directed RNA,Polymerase, RNA,Polymerases, DNA-Dependent RNA,Polymerases, DNA-Directed RNA,Polymerases, RNA,RNA Polymerase, DNA-Directed,RNA Polymerases, DNA-Dependent,RNA Polymerases, DNA-Directed
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D013194 Staining and Labeling The marking of biological material with a dye or other reagent for the purpose of identifying and quantitating components of tissues, cells or their extracts. Histological Labeling,Staining,Histological Labelings,Labeling and Staining,Labeling, Histological,Labelings, Histological,Stainings
D017790 Closterovirus A genus of plant viruses in the family CLOSTEROVIRIDAE containing highly flexuous filaments. Some members are important pathogens of crop plants. Natural vectors include APHIDS, whiteflies, and mealybugs. The type species is Beet yellows virus. Beet yellow stunt virus,Beet yellows virus

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