Molecular techniques should now replace cell culture in diagnostic virology laboratories

Carman, Bill

doi:10.1002/rmv.334

Cited by 22 publications

(16 citation statements)

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“…Indeed, molecular diagnostics are deemed superior to bacterial culture techniques or serological diagnostics (2)(3)(4). It has even been suggested to entirely eliminate the old methods in order to streamline centralized laboratories for molecular diagnostics (5)(6)(7).…”

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confidence: 99%

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

et al. 2013

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Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms. Syndromic panels for infectious and emerging infectious diseases have been suggested to be of value in point-of-care (POC) diagnostics for developing countries and for biodefense (1). Since the introduction of molecular diagnostics and in particular real-time PCR, ample proof of its sensitivity and specificity has been generated. Indeed, molecular diagnostics are deemed superior to bacterial culture techniques or serological diagnostics (2-4). It has even been suggested to entirely eliminate the old methods in order to streamline centralized laboratories for molecular diagnostics (5-7).In recent years alternative isothermal amplification methods which can be categorized into (i) T7 promoter-driven amplifications (transcription-mediated amplification [TMA], nucleic acid sequence-based amplification [NASBA], and single primer isothermal amplification [SPIA]), (ii) strand displacement methods (strand displacement amplification [SDA], loop-mediated isothermal amplification [LAMP], and smart amplification [SmartAmp]), (iii) helicase-dependent amplification (HDA), (iv) recombinase polymerase amplification (RPA), and (v) rolling-circle amplification (RCA) methods (8-12) have been developed. Some were purposely designed for isothermal amplification starting from RNA (TMA, NASBA, and SPIA), whereas others initially targeted DNA (SDA, LAMP, HDA, RPA, and RCA) and were only later adapted for RNA targets. Nonspecific intercalating fluorophores or fluorescent primers have been used for real-time detection in LAMP, SDA, HDA, and RCA, and specific detection probe formats have been developed for NASBA, RCA, HDA,.In isothermal and exponential RPA, the phage recombinase UvsX and its cofactor UvsY form a nucleoprotein complex with oligonucleotide primers to scan for homologous sequences in a DNA template. Recognition of a specific homologous sequence leads to the initiation of strand invasion of the com...

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confidence: 99%

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

et al. 2013

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“…Furthermore the explosive growth of PCR based diagnostics has led to the introduction of many different techniques that allow convenit detection of PCR products, especially nested PCR diagnostic methods. These produce nucleic acid frgments specific to the viruses studied that can be used for characterization and identification [12,2]. It is more advantageous to use ISH to detect viral DNA in tissues or cells than to use histological staining or electron microscopy.…”

Section: Discussionmentioning

confidence: 99%

“…After deparaffinization and treatment for 20 min at 37°C with 40 µg/ml proteinase K (Roche), the sections were post-fixed for 10 min at room temperature with 4% paraformaldehyde and then washed with PBS and DEPC-treated dH 2 O and covered with 30 µl amplification mixture which contained 4.5 mM MgCl 2 , 10 µM digoxigenin-11dUTP (Roche), 190 µM dTTP, 200 µM dATP, dGTP, and dCTP, 1× PCR buffer, 1 µM of each primer C500-3 and C500-4, 1× self Seal reagent (MJ Research, Waltham, MA, U.S.A.), 0.1% BSA, 5 U Ampli Taq Gold (Roche). They were covered with sterile cover slip and placed in the in situ PCR Thermal Cycler (Gene Amp In Situ PCR System 1,000, PerkinElmer, Foster, CA, U.S.A.).…”

Section: Preparation Of Infected Cellsmentioning

confidence: 99%

Development of in situ Nest PCR and Comparison of Five Molecular Biological Diagnostic Methods for the Detection of Intracellular Viral DNAs in Paraffin Sections.

Kim

2003

The Journal of Veterinary Medical Science

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ABSTRACT. Nest polymerase chain reaction (PCR), in situ hybridization (ISH), in situ PCR, in situ PCR/hybridization (PCR-ISH) and in situ nest PCR were compared for the detection and localization of intracellular viral DNAs in paraffin sections. MDBK cells were infected with alcelapine herpesvirus 1 ranging from 10 1 to 10 5 50% tissue culture infected doses (TCID 50 ), incubated 18 hr, then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to nest PCR, ISH, in situ PCR, PCR-ISH and in situ nest PCR using specific oligonucleotide primers or probes directed against the viral open reading frame 50. In situ nest PCR and nest PCR were found to be capable of detecting the viral DNA in the cells infected with the lowest virus titer. As compared with other molecular biological methods for the detection of the virus, in situ nest PCR was found to be more sensitive than ISH, in situ PCR and PCR-ISH. In situ nest PCR has wide applications for sensitive localization of low copy viral sequences within cells to investigate the role of viruses in a variety of clinical conditions. KEY WORDS: in situ hybridization, in situ PCR, in situ PCR/hybridization, in situ nest PCR.

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“…Some viruses cannot be grown in standard cultures and sensitivity is often too low to reliably detect low amounts of virus (as with cerebrospinal fluid (CSF) in HSV encephalitis). The arrival of the polymerase chain reaction (PCR) in laboratories has revolutionised the impact of diagnostic virology in clinical practice (Carman, 2001). This technique is a highly sensitive and specific method for the detection of viral nucleic acid, and can be adapted to detect practically any virus in any body fluid or tissue.…”

Section: Confirm the Diagnosismentioning

confidence: 99%

Strategies for the Rational Use of Antivirals

Waugh

Carman

2005

Antibiotic Policies

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Molecular techniques should now replace cell culture in diagnostic virology laboratories

Cited by 22 publications

References 11 publications

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Development of a Panel of Recombinase Polymerase Amplification Assays for Detection of Biothreat Agents

Development of in situ Nest PCR and Comparison of Five Molecular Biological Diagnostic Methods for the Detection of Intracellular Viral DNAs in Paraffin Sections.

Strategies for the Rational Use of Antivirals

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