Alternative Molecular Tests for Virological Diagnosis

Sidoti, Francesca; Bergallo, Massimiliano; Costa, Cristina; Cavallo, Rossana

doi:10.1007/s12033-012-9533-8

Cited by 21 publications

(17 citation statements)

References 101 publications

(99 reference statements)

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“…These new techniques do not require thermal cycler and can be performed by using a heating block and/or water bath. The main isothermal methods include loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA) [33].…”

Section: Isothermal Methodsmentioning

confidence: 99%

Application of Molecular Diagnostic Techniques for Viral Testing

Cobo¹

2012

TOVJ

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Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.

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Section: Isothermal Methodsmentioning

confidence: 99%

Application of Molecular Diagnostic Techniques for Viral Testing

Cobo¹

2012

TOVJ

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“…These samples were prescreened (except for samples 201000496, Tx09-58, and Tx08-25) for the presence of HPV using a multiplex PCR (unpublished data). Totals of 30,17,8,12, and 10 HPV-infected samples were tested and found to be positive for HPV using endpoint RT-PCR, TaqMan RT-qPCR, SYBR green RT-qPCR, RT-HDA, and Razor Ex (Table 1). No contradictory results were obtained with any of the methods (Table 1).…”

Section: Fig 2 Location Of Hpv Primers On Nucleoprotein Gene (Genbankmentioning

confidence: 99%

“…The use of isothermal virus detection methods is gaining wide popularity because they provide an alternative method for nucleic acid amplification with no need for sophisticated and expensive thermocyclers and can be performed using a heat block or water bath at just two constant temperatures (17,18). Loop-mediated isothermal amplification has been reported for plant virus detection (19)(20)(21)(22), However, there are only a few reports of the use of RT-helicase dependent amplification (RT-HDA) detection with plant-pathogenic viruses (23) and other viruses (24,25), and these applications required the use of a primer with a T m of 68°C.…”

mentioning

confidence: 99%

Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

Arif

Aguilar-Moreno

Wayadande

et al. 2014

Appl Environ Microbiol

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A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods-real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)-for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5= terminus, raising the primers' melting temperature (T m ; ca. 10°C) to make them compatible with RT-HDA (required optimal T m ‫؍‬ 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.

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“…An accurate discrimination between various Phytophthora species was successfully realized by polymerase chain reaction (PCR) 5 . Nevertheless, their application in the field is hampered due to the need for thermal cycling instruments [7][8][9][10][11][12] .…”

Section: Introductionmentioning

confidence: 99%

“…An important step towards on-site detection of regulated Phytophthora species is provided by isothermal nucleic acid amplification techniques [7][8][9][10][11][12] . Recently, several articles highlight the loop-mediated amplification (LAMP) for a DNA-based Phytophthora specification [13][14][15] .…”

Section: Introductionmentioning

confidence: 99%

Towards on-site testing of Phytophthora species

et al. 2015

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Rapid detection and accurate identification of plant pathogens in the field is an ongoing challenge. In this study, we report for the first time on the development of a helicasedependent isothermal amplification (HDA) in combination with on-chip hybridization for the detection of selected Phytophthora species. The HDA approach allows the efficient amplification of the yeast GTP-binding protein (Ypt1) target gene region at one constant temperature in a miniaturized heating device. The assay's specificity was determined by onchip DNA hybridization and subsequent silver nanoparticle deposition. The silver deposits serve as stable endpoint signals that enable the visual as well as the electrical readout. Our promising results point into the direction of a near future on-site application of the combined techniques for a reliable detection of Phytophthora species.

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Alternative Molecular Tests for Virological Diagnosis

Cited by 21 publications

References 101 publications

Application of Molecular Diagnostic Techniques for Viral Testing

Application of Molecular Diagnostic Techniques for Viral Testing

Primer Modification Improves Rapid and Sensitive In Vitro and Field-Deployable Assays for Detection of High Plains Virus Variants

Towards on-site testing of Phytophthora species

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