Using the LN34 Pan-Lyssavirus Real-Time RT-PCR Assay for Rabies Diagnosis and Rapid Genetic Typing from Formalin-Fixed Human Brain Tissue

Human rabies post mortem diagnostic samples are often preserved in formalin. While immunohistochemistry (IHC) has been routinely used for rabies antigen detection in formalin-fixed tissue, the formalin fixation process causes nucleic acid fragmentation that may affect PCR amplification. This study reports the diagnosis of rabies in an individual from the Dominican Republic using both IHC and the LN34 pan-lyssavirus real-time RT-PCR assay on formalin-fixed brain tissue. The LN34 assay generates a 165 bp amplicon and demonstrated higher sensitivity than traditional PCR. Multiple efforts to amplify nucleic acid fragments larger than 300 bp using conventional PCR were unsuccessful, probably due to RNA fragmentation. Sequences generated from the LN34 amplicon linked the case to the rabies virus (RABV) strain circulating in the Ouest Department of Haiti to the border region between Haiti and the Dominican Republic. Direct sequencing of the LN34 amplicon allowed rapid and low-cost rabies genetic typing.

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Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.287 ◽

2021 ◽

Vol 156 (Supplement_1) ◽

pp. S134-S135

Author(s):

T Berent ◽

T Rothstein ◽

S Buckwalter ◽

R Patel

Keyword(s):

Real Time ◽

Whole Blood ◽

New Technologies ◽

Limit Of Detection ◽

Rt Pcr ◽

Bartonella Species ◽

Pcr Assay ◽

Fixed Tissue ◽

Hybridization Probes ◽

Ffpe Samples

Abstract Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

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High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

Clinical Chemistry ◽

10.1373/clinchem.2006.070987 ◽

2007 ◽

Vol 53 (1) ◽

pp. 104-110 ◽

Cited By ~ 46

Author(s):

Melanie Störmer ◽

Knut Kleesiek ◽

Jens Dreier

Keyword(s):

Detection Limit ◽

Nucleic Acid ◽

Nucleic Acids ◽

Real Time ◽

High Volume ◽

Magnetic Bead ◽

Locked Nucleic Acid ◽

Rt Pcr ◽

Pcr Assay ◽

Probe System

Abstract Background: Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid–binding matrices. We adapted this technology with a broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products. Methods: We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresis-derived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primer–probe system and locked nucleic acid technology. Coamplification of human β2-microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence. Results: For automated magnetic bead–based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 × 103 colony-forming units (CFU)/L for S. epidermidis and 22 × 103 CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid contamination of reagents or externally during testing of 1030 PCs. Conclusions: High-volume nucleic acid extraction improved the detection limit of the assay. The improvement of the primer–probe system and the integration of an IC make the RT-PCR assay appropriate for bacteria screening of platelets.

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A35 The first laboratory confirmation of chikungunya outbreak in Ethiopia

Virus Evolution ◽

10.1093/ve/vez002.034 ◽

2019 ◽

Vol 5 (Supplement_1) ◽

Author(s):

Mesfin Mengesha Tsegaye ◽

Aadamu Tayachew ◽

Desalegn Belay ◽

Abebe Alemu ◽

Berhane Beyene

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Laboratory Investigation ◽

Emergency Preparedness ◽

Zika Virus ◽

Chikungunya Virus ◽

Febrile Illness ◽

Rt Pcr ◽

Pcr Assay ◽

Serum Samples

Abstract Chikungunya is a viral disease (genus Alphavirus) which is transmitted to humans by infected mosquitoes—including Aedes aegypti and A. albopictus. An outbreak of febrile illness, suspected to have been caused by chikungunya, was reported in June 2016 from Dolloado district, Suuf Kebele, in the Somalia regional state of Ethiopia that borders the Mandera county of Kenya where a confirmed chikungunya outbreak was ongoing. Laboratory investigation was carried out to confirm if the outbreak in Ethiopia was caused by Chikungunya virus. Ten serum samples were collected from suspected patients visiting a health center in Suuf Kebel, who were then sent to the Nation laboratory in Ethiopian Public Health Institute. RNA was extracted from the serum samples using QIAgene RNA Mini kit, and PCR detection of dengue, chikungunya, and Zika virus nucleic acid was done using Trioplex Real-time RT-PCR Assay following the protocol from the Center for Disease Control (CDC). The Trioplex Real-time RT-PCR assay, for detection and differentiation of RNA from dengue, Chikungunya and Zika, was provided by CDC as part of the zika emergency preparedness effort. Of the nine samples tested, eight (88.88%) were found to be positive for chikungunya virus nucleic acid but negative for dengue and Zika virus nucleic acids. The median age of the affected sampled patients was 40 years, and males appear to be more affected (66.6% of sampled patients). The laboratory investigation confirmed that the outbreak was caused by chikungunya virus. Even though further molecular characterization of the positive isolates will provide more information as to the circulating genotypes and elucidate the origin of the outbreak virus, it is also possible to assume that the outbreak was an extension of the outbreak in neighboring countries in Kenya and, therefore, warrants that cross-border integration efforts to control chikungunya should be implemented by the concerned countries.

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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Development and application of a real-time RT-PCR assay to rapidly detect H2 subtype avian influenza A viruses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638721994810 ◽

2021 ◽

pp. 104063872199481

Author(s):

Yixin Xiao ◽

Fan Yang ◽

Fumin Liu ◽

Hangping Yao ◽

Nanping Wu ◽

...

Keyword(s):

Avian Influenza ◽

Real Time ◽

Influenza A ◽

Minor Groove Binder ◽

Rt Pcr ◽

Influenza A Viruses ◽

Pcr Assay ◽

Infectious Dose ◽

The Matrix ◽

Taqman Minor Groove Binder

The H2 subtypes of avian influenza A viruses (avian IAVs) have been circulating in poultry, and they have the potential to infect humans. Therefore, establishing a method to quickly detect this subtype is pivotal. We developed a TaqMan minor groove binder real-time RT-PCR assay that involved probes and primers based on conserved sequences of the matrix and hemagglutinin genes. The detection limit of this assay was as low as one 50% egg infectious dose (EID50)/mL per reaction. This assay is specific, sensitive, and rapid for detecting avian IAV H2 subtypes.

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A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

Journal of Virological Methods ◽

10.1016/j.jviromet.2013.02.004 ◽

2013 ◽

Vol 189 (2) ◽

pp. 277-282 ◽

Cited By ~ 11

Author(s):

Yong Yan ◽

Heng-hui Wang ◽

Lei Gao ◽

Ji-mei Ji ◽

Zhi-jie Ge ◽

...

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Human Norovirus ◽

One Step

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Multicenter evaluation of the ENTEROVIRUS R-gene™ real-time RT-PCR assay for the detection of enteroviruses in clinical specimens

Journal of Clinical Virology ◽

10.1016/j.jcv.2009.09.033 ◽

2010 ◽

Vol 47 (1) ◽

pp. 54-59 ◽

Cited By ~ 16

Author(s):

Sylvie Pillet ◽

Geneviève Billaud ◽

Shabir Omar ◽

Bruno Lina ◽

Bruno Pozzetto ◽

...

Keyword(s):

Real Time ◽

R Gene ◽

Rt Pcr ◽

Pcr Assay ◽

Clinical Specimens

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Performance characteristics of a real-time RT-PCR assay for quantification of hepatitis C virus RNA in patients with genotype 1 and 2 infections

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2008.082 ◽

2008 ◽

Vol 46 (4) ◽

Cited By ~ 2

Author(s):

Jee-Fu Huang ◽

Chia-Yen Dai ◽

Ya-Yun Lin ◽

Ming-Lung Yu ◽

Shu-Fen Liu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Real Time ◽

Performance Characteristics ◽

Genotype 1 ◽

Rt Pcr ◽

Hepatitis C Virus Rna ◽

Pcr Assay ◽

Virus Rna

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Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽

10.1094/pdis-03-12-0283-re ◽

2013 ◽

Vol 97 (5) ◽

pp. 641-644 ◽

Cited By ~ 17

Author(s):

Manphool S. Fageria ◽

Mathuresh Singh ◽

Upeksha Nanayakkara ◽

Yvan Pelletier ◽

Xianzhou Nie ◽

...

Keyword(s):

Real Time ◽

Potato Virus ◽

Potato Virus Y ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Pcr Assay ◽

Current Season ◽

Seed Market ◽

Polymerase Chain ◽

Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

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