scholarly journals Multiplex Nested Reverse Transcription-Polymerase Chain Reaction in a Single Tube for Sensitive and Simultaneous Detection of Four RNA Viruses and Pseudomonas savastanoi pv. savastanoi in Olive Trees

2003 ◽  
Vol 93 (3) ◽  
pp. 286-292 ◽  
Author(s):  
Edson Bertolini ◽  
Antonio Olmos ◽  
María M. López ◽  
Mariano Cambra
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Nested Pcr ◽  
Rna Viruses ◽  
Colorimetric Detection ◽  
Simultaneous Detection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Olive Trees

A multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) in a single closed tube was developed for the simultaneous detection of four RNA viruses: Cucumber mosaic virus, Cherry leaf roll virus, Strawberry latent ringspot virus, and Arabis mosaic virus, and the bacterium Pseudomonas savastanoi pv. savastanoi. The method enabled, for the first time, the sensitive and simultaneous detection of RNA and DNA targets from plant viruses and a bacterium, saving time, decreasing risks of contamination, and reducing costs compared with conventional monospecific nested amplifications. The method was successfully coupled with colorimetric detection of amplicons using specific oligoprobes to simplify routine detection. Two hundred forty-five olive trees from 15 different cultivars were analyzed by multiplex RT-nested PCR coupled with colorimetric detection. Multiplex nested RT-PCR for viral detection increased the identification of positive trees by 8.1%. An uneven distribution of the viruses was observed in the infected trees. The bacterium was detected in 28.7% of the analyzed trees by the developed multiplex nested method and by a nested PCR previously developed. This powerful methodology could be applied to other models for the detection of several pathogens in a single assay.

A Multiplex Reverse Transcription Polymerase Chain Reaction Method for the Detection of Foodborne Viruses†

1999 ◽  
Vol 62 (10) ◽  
pp. 1210-1214 ◽  
Author(s):  
SORAYA I. ROSENFIELD ◽  
LEE-ANN JAYKUS
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Simultaneous Detection ◽  
Polymerase Chain Reaction Method ◽  
Human Enterovirus ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

A multiplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the simultaneous detection of the human enteroviruses, hepatitis A virus (HAV) and Norwalk virus (NV). Poliovirus type 1 (PV1) was chosen as a model for the human enterovirus group. Three different sets of primers were used to produce three size-specific amplicons of 435 bp, 270 bp, and 192 bp for PV1, NV, and HAV, respectively. RT-PCR products were separated by agarose gel electrophoresis, and amplicon identity was confirmed by Southern transfer followed by DNA hybridization using nonradio-active, digoxigenin-labeled internal probes. When tested on mixed, purified virus suspensions, the multiplex method achieved detection limits of ≤1 infectious unit (PV1 and HAV) or RT-PCR-amplifiable unit (NV) for all viruses. With further streamlining efforts such as single tube amplification and liquid hybridization, multiplex PCR offers advantages over cell culture methodology and monoplex PCR because it allows for rapid and cost-effective detection of several human enteric viruses in a single reaction tube.

scholarly journals Simultaneous Detection of the Defoliating and Nondefoliating Verticillium dahliae Pathotypes in Infected Olive Plants by Duplex, Nested Polymerase Chain Reaction

Plant Disease ◽  
2003 ◽  
Vol 87 (12) ◽  
pp. 1487-1494 ◽  
Author(s):  
J. Mercado-Blanco ◽  
D. Rodríguez-Jurado ◽  
S. Parrilla-Araujo ◽  
R. M. Jiménez-Díaz
Keyword(s):  
Polymerase Chain Reaction ◽  
Verticillium Dahliae ◽  
Nested Pcr ◽  
Simultaneous Detection ◽  
Specific Marker ◽  
In Planta ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Polymerase Chain ◽  
Olive Trees

Pathogen-free certified planting material and accurate detection of Verticillium dahliae pathotypes infecting the plant are key components of successful management of Verticillium wilt of olive. Use of a nested-polymerase chain reaction (PCR) procedure developed in earlier studies for in planta detection of the defoliating (D) and nondefoliating (ND) V. dahliae pathotypes resulted in ambiguous detection of the pathogen in some cases, due to heterologous amplification of the D-associated marker in ND-infected olive plants. In the present study, an improved procedure was developed that eliminates ambiguity and reduces time and cost for detection of D and ND V. dahliae in olive. The improved procedure is based on the simultaneous amplification of both an ND- and a new D-specific marker by means of duplex, nested PCR. The procedure was effective in the rapid and unequivocal detection of the D and ND V. dahliae in both artificially inoculated, own-rooted olive plants and naturally infected adult olive trees of different cultivar, age, and growing conditions. Furthermore, the duplex, nested-PCR procedure detected simultaneously the D and ND pathotypes in adult olive trees naturally infected by both pathotypes and in young olive plants that were double-inoculated with D and ND isolates under controlled conditions.

scholarly journals A One-Step Reverse Transcription-Polymerase Chain Reaction System for the Simultaneous Detection and Identification of Multiple Tospovirus Infections

2005 ◽  
Vol 95 (2) ◽  
pp. 166-171 ◽  
Author(s):  
Hiroyuki Uga ◽  
Shinya Tsuda
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Simultaneous Detection ◽  
Yellow Spot ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Spot Virus ◽  
Detection And Identification ◽  
One Step ◽  
Polymerase Chain

A one-step reverse transcription-polymerase chain reaction (RT-PCR) method has been developed for the simultaneous detection and identification of multiple tospoviruses that infect plants. The RT-PCR system is composed of six primers in a single tube: a universal degenerate primer and five virus species-specific primers. Amplifications resulted in an 848-bp PCR product for Watermelon silver mottle virus, 709-bp for Tomato spotted wilt virus, 589-bp for Impatiens necrotic spot virus, 511-bp for Melon yellow spot virus, and a 459-bp amplicon for Iris yellow spot virus. This system enables the simultaneous detection of at least three types of tospovirus infections, in addition to their species identities, from five possible tospoviruses studied, on the basis of their S RNA combinations. This multiplex RT-PCR system was applied to the detection of tospovirus in ornamental crops cultivated in fields and shows potential for epidemiological studies.

scholarly journals Simultaneous Detection of Nine Grapevine Viruses by Multiplex Reverse Transcription-Polymerase Chain Reaction with Coamplification of a Plant RNA as Internal Control

2006 ◽  
Vol 96 (11) ◽  
pp. 1223-1229 ◽  
Author(s):  
Giorgio Gambino ◽  
Ivana Gribaudo
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Control ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Simultaneous Detection ◽  
Grapevine Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Arabis Mosaic Virus ◽  
Polymerase Chain

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for simultaneous detection of nine grapevine viruses: Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Rupestris stem pitting-associated virus, Grapevine fleck virus, Grapevine leafroll-associated virus-1, -2, and -3, in combination with a plant RNA internal control used as an indicator of the effectiveness of RNA extraction and RT-PCR. Primers were designed from conserved regions of each virus and their specificity was confirmed by sequencing PCR products. Two plant total RNA extraction methods (silica capture and modified RNeasy method) and two RT-PCR systems (onestep and two-step) were evaluated to develop a reliable protocol for mRT-PCR. One to nine fragments specific for the viruses were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel electrophoresis. In the two-step mRT-PCR, the detection limits were 10-3 or 10-4 extract dilutions, depending on the virus. Leaves, phloem from dormant cuttings, and in vitro plantlets from 103 naturally infected and healthy grapevines were analyzed. The mRT-PCR provided a reliable and rapid method for detecting grapevine viruses from a large number of samples.

scholarly journals The Epidemiological Characteristics of the Korean Bat Paramyxovirus between 2016 and 2019

2020 ◽  
Vol 8 (6) ◽  
pp. 844
Author(s):  
Seong Sik Jang ◽  
Ji Yeong Noh ◽  
Van Thi Lo ◽  
Yong Gun Choi ◽  
Sun-Woo Yoon ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Nested Pcr ◽  
Human Pathogens ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
Rdrp Region ◽  
Polymerase Chain ◽  
Epidemiological Characteristics

Bats are considered reservoirs of severe emerging human pathogens. Notably, bats host major mammalian paramyxoviruses from the family Paramyxoviridae, order Mononegavirales. In this study, paramyxoviruses were investigated by reverse transcription semi-nested polymerase chain reaction (RT-semi-nested PCR) and reverse transcription polymerase chain reaction (RT-PCR), based on the RT-semi-nested PCR using the consensus paramyxovirus primers targeting the RNA dependent-RNA-polymerase (RdRp) region. In addition, RT-PCR was performed using newly designed primers targeting regions of the fusion protein (F) and hemagglutinin-neuraminidase (HN). The dominant bat species in the collection site of paramyxoviruses were Miniopterus schreibersii, Myotis macrodactylus, Myotis petax, and Rhinolophus ferrumequinum. Paramyxoviruses were detected in four samples in 2016 and six in 2019. Meanwhile, in samples collected in 2017 and 2018, no paramyxoviruses were detected. Phylogenetic analysis based on the partial nucleotide sequences of RdRp, F, and HN proteins suggested that the viruses belonged to the proposed genus Shaanvirus. In conclusion, this study revealed that bat paramyxoviruses in Korea belonged to a single genus and circulated sporadically in several provinces, including Chungbuk, Gangwon, Jeju, and Jeonnam.

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