Development of a Tandem Repeat-Based Polymerase Chain Displacement Reaction Method for Highly Sensitive Detection of 'Candidatus Liberibacter asiaticus'. 2018

Binghai Lou, and Yaqin Song, and Moytri RoyChowdhury, and Chongling Deng, and Ying Niu, and Qijun Fan, and Yan Tang, and Changyong Zhou
First author: College of Plant Protection, Southwest University, Chongqing 400715, P.R. China; first, second, fourth, fifth, sixth, and seventh authors: Guangxi Key Laboratory of Citrus Biology, Guangxi Academy of Specialty Crops, Guilin, Guangxi 541004, P.R. China; third author: Department of Biological Sciences, Idaho State University, 650 Memorial Dr., Pocatello 83201; and eighth author: Citrus Research Institute, Southwest University, Chongqing 400715, P.R. China.

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of 'Candidatus Liberibacter asiaticus', a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of 'Ca. L. asiaticus' with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of 'Ca. L. asiaticus'.

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
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D012230 Rhizobiaceae A family of gram-negative bacteria which are saprophytes, symbionts, or plant pathogens. Bradyrhizobium lupini,Neorhizobium galegae,Rhizobium galegae,Rhizobium lupini
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
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
D020080 Tandem Repeat Sequences Copies of DNA sequences which lie adjacent to each other in the same orientation (direct tandem repeats) or in the opposite direction to each other (INVERTED TANDEM REPEATS). Direct Tandem Repeats,Tandem Repeat,Tandem Repeats,Direct Tandem Repeat,Repeat Sequence, Tandem,Repeat Sequences, Tandem,Repeat, Direct Tandem,Repeat, Tandem,Repeats, Direct Tandem,Repeats, Tandem,Sequence, Tandem Repeat,Sequences, Tandem Repeat,Tandem Repeat Sequence,Tandem Repeat, Direct,Tandem Repeats, Direct

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