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Since 2013, group A rotavirus strains characterized as novel DS-1-like intergenogroup reassortant “equine-like G3” strains have emerged and spread across 5 continents among human populations in at least 14 countries. Here, we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 227 to 2.3 × 10(9) copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like intergenogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost. It will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

In 2009, three children were hospitalized in Rochester, NY, with sequence-confirmed G8P[4] rotavirus gastroenteritis—the first U.S. detection of this uncommon strain more typically found in Africa. Continued monitoring of G8P[4] and other rotavirus genotypes not represented in current vaccines is essential to assess whether vaccination will result in an increase in prevalence of these strains.

Definitive identification of Brucella species remains a challenge due to the high degree of genetic homology shared within the genus. We report the development of a molecular technique which utilizes real-time PCR followed by high-resolution melt (HRM) curve analysis to reliably type members of this genus. Using a panel of seven primer sets, we tested 153 Brucella spp. isolates with >99% accuracy compared to traditional techniques. This assay provides a useful diagnostic tool that can rapidly type Brucella isolates and has the potential to detect novel species. This approach may also prove helpful for clinical, epidemiological and veterinary investigations.

A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [Tm], 56.40°C), monkeypox virus (Tm, 56.24°C), and vaccinia virus (Tm, 56.72°C), including the Dryvax vaccine strain, from smallpox virus (Tm, 62.45°C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR. Smallpox virus is a major threat as an agent of bioterrorism. Clinical infection is characterized by an incubation period of 12 to 14 days (range, 7 to 17 days), followed by the onset of high fever, malaise, and prostration, often with severe headache, nausea, and vomiting (6). A rash that progresses from the papular to vesicular stages and eventually to the pustular stage generally appears by the third day of illness and initially appears in the mouth on the oral mucosa, on the face, and on the head and then spreads to the limbs and trunk of the body. Smallpox patients can transmit the virus during the first week, when the lesions in the oral mucosa ulcerate and seed the saliva with smallpox virus (6, 21, 23, 27, 28). The disease causes severe morbidity and can lead to a mortality rate of 30% among the victims. The family Poxviridae and the subfamily Chordopoxvirinae (poxviruses of vertebrates) contain eight genera; importantly, smallpox virus and the closely related cowpox virus, vaccinia virus, and monkeypox virus (the vaccine strain used for immunization against smallpox) can infect humans and are classified in the single genus Orthopoxvirus. These DNA viruses are large brick-shaped particles that vary in size (length, 220 to 450 nm; width, 140 to 260 nm); the genome of smallpox virus consists of 186 kb of linear double-stranded DNA (19). Two molecular amplification tests have previously been described for the detection and discrimination of viruses within the genus Orthopoxvirus; however, labor-intensive conventional PCR with consensus or sequence-specific primers and subsequent digestion of amplicons with several restriction endonucleases followed by gel electrophoresis were required to identify the specific viruses within this group (20, 25). Recently, reference strains of smallpox virus and scabs from patients (Russian archived specimens) with this infection were detected in a 6-h test by a homemade PCR amplification and detection system based on oligonucleotide microchip technology (17). We describe a rapid (3-h [for nucleic acid extraction and amplification]) real-time PCR (LightCycler; Roche Applied Science, Indianapolis, Ind.) that detects smallpox virus and that differentiates the DNA of this agent from the DNAs of other viruses classified within the genus Orthopoxvirus.

A real-time quantitative reverse transcription-PCR (qRT-PCR) assay using the recombinant thermostable Thermus thermophilus (r Tth ) enzyme was developed to detect and quantify rotavirus A (RVA). By using r Tth polymerase, significant improvement was achieved over the existing real-time RT-PCR assays, which require denaturation of the RVA double-stranded RNA (dsRNA) prior to assay setup. Using a dsRNA transcript for segment 7, which encodes the assay target NSP3 gene, the limit of detection for the improved assay was calculated to be approximately 1 genome copy per reaction. The NSP3 qRT-PCR assay was validated using a panel of 1,906 stool samples, 23 reference RVA strains, and 14 nontarget enteric virus samples. The assay detected a diverse number of RVA genotypes and did not detect other enteric viruses, demonstrating analytical sensitivity and specificity for RVA in testing stool samples. A XenoRNA internal process control was introduced and detected in a multiplexed qRT-PCR format. Because it does not require an antecedent dsRNA denaturation step, this assay reduces the possibility of sample cross-contamination and requires less hands-on time than other published qRT-PCR protocols for RVA detection.

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus , B. melitensis , and B. suis . Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella .