Biomaterial Database

Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid. 2014

Lambros C Papayiannis

Agricultural Research Institute (ARI), P.O. Box 22016, Nicosia 1516, Cyprus. Electronic address: l.papayiannis@ari.gov.cy.

Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.

UI	MeSH Term	Description	Entries
D010935	Plant Diseases	Diseases of plants.	Disease, Plant,Diseases, Plant,Plant Disease
D002957	Citrus	A plant genus of the family RUTACEAE. They bear the familiar citrus fruits including oranges, grapefruit, lemons, and limes. There are many hybrids which makes the nomenclature confusing.	Citron Tree,Fruit, Citrus,Lemon Tree,Orange Tree, Bitter,Orange Tree, Mandarin,Orange Tree, Seville,Orange Tree, Sour,Pomelo Tree,Pummelo Tree,Tangerine Tree,Citrus Fruit,Citrus aurantium,Citrus bergamia,Citrus grandis,Citrus hystrix,Citrus limon,Citrus maxima,Citrus medica,Citrus reticulata,Kaffir Lime,Bitter Orange Tree,Bitter Orange Trees,Citron Trees,Citrus aurantiums,Citrus bergamias,Citrus grandi,Citrus hystrices,Citrus medicas,Citrus reticulatas,Lemon Trees,Lime, Kaffir,Mandarin Orange Tree,Mandarin Orange Trees,Orange Trees, Bitter,Orange Trees, Mandarin,Orange Trees, Seville,Orange Trees, Sour,Pomelo Trees,Pummelo Trees,Seville Orange Tree,Seville Orange Trees,Sour Orange Tree,Sour Orange Trees,Tangerine Trees,Tree, Bitter Orange,Tree, Citron,Tree, Lemon,Tree, Mandarin Orange,Tree, Pomelo,Tree, Pummelo,Tree, Seville Orange,Tree, Sour Orange,Tree, Tangerine,Trees, Bitter Orange,Trees, Citron,Trees, Lemon,Trees, Mandarin Orange,Trees, Pomelo,Trees, Pummelo,Trees, Seville Orange,Trees, Sour Orange,Trees, Tangerine,aurantium, Citrus,bergamia, Citrus,hystrices, Citrus,maxima, Citrus,medicas, Citrus,reticulata, Citrus
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
D014772	Viroids	A group of pathogens comprising the smallest known agents of infectious disease. They are unencapsulated and are capable of replicating autonomously in susceptible cells. Positively identified viroids composed of single-stranded RNA have been isolated from higher plants, but the existence of DNA viroids pathogenic to animals is suspected.	Viroid
D060888	Real-Time Polymerase Chain Reaction	Methods used for detecting the amplified DNA products from the polymerase chain reaction as they accumulate instead of at the end of the reaction.	Kinetic Polymerase Chain Reaction,Quantitative Real-Time PCR,Quantitative Real-Time Polymerase Chain Reaction,Real-Time PCR,PCR, Quantitative Real-Time,PCR, Real-Time,PCRs, Quantitative Real-Time,PCRs, Real-Time,Quantitative Real Time PCR,Quantitative Real Time Polymerase Chain Reaction,Quantitative Real-Time PCRs,Real Time PCR,Real Time Polymerase Chain Reaction,Real-Time PCR, Quantitative,Real-Time PCRs,Real-Time PCRs, Quantitative
D020133	Reverse Transcriptase Polymerase Chain Reaction	A variation of the PCR technique in which cDNA is made from RNA via reverse transcription. The resultant cDNA is then amplified using standard PCR protocols.	Polymerase Chain Reaction, Reverse Transcriptase,Reverse Transcriptase PCR,PCR, Reverse Transcriptase,Transcriptase PCR, Reverse
D027582	Humulus	A plant genus in the CANNABACEAE family. Best known for the buds of Humulus lupulus L. used in BEER.	Hops,Humulus lupulus