A Multiplex PCR/LDR Assay for Viral Agents of Diarrhea with the Capacity to Genotype Rotavirus.

Rotavirus and noroviruses are major causes of diarrhea. Variable rotavirus vaccination efficacy in Africa and Asia is multifactorial, including the diversity of circulating strains and viral co-infection. We describe a multiplexed assay that detects and genotypes viruses from stool specimens. It includes a one-step reverse transcriptase PCR reaction, a ligase detection reaction (LDR), then hybridization of fluorescent products to micro-beads. In clinical samples it detects rotavirus, caliciviruses (sapovirus and norovirus), mixed infections, and genotypes or genogroups of rotaviruses and noroviruses, respectively. The assay also has the capacity to detect hepatitis A. The assay was validated on reference isolates and 296 stool specimens from the US and Ghana. The assay was 97% sensitive and 100% specific. The genogroup was concordant in 100% of norovirus, and the genotype in 91% and 89% of rotavirus G- and P-types, respectively. Two rare rotavirus strains, G6P[6] and G6P[8], were detected in stool specimens from Ghana. The high-throughput assay is sensitive, specific, and may be of utility in the epidemiological surveillance for rare and emerging viral strains post-rotavirus vaccine implementation.

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus.

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

The comet assay: genotoxic damage or nuclear fragmentation?

The single cell gel electrophoresis (SCGE) or comet assay is based on the assumption that comet images result from genotoxic damage that ultimately generate DNA single- or double-strand breaks. A criticism of the assay is that some or all of the comet images may be the result of apoptosis-mediated nuclear fragmentation. The objective of this study was to determine if mutagen-induced DNA damage leading to strand breakage observed in the SCGE assay was repairable or was due to nonrepairable nuclear fragmentation. Chinese hamster ovary cells were treated with ethylmethanesulfonate, 2-acetoxyacetylaminofluorene, or H(2)O(2). These mutagens induce genetic damage by different molecular mechanisms. One group of SCGE slides was prepared immediately after treatment, while parallel treated cultures were repeatedly washed and allowed to undergo liquid holding recovery for DNA repair. It was hypothesized that cells with genotoxic damage can repair their genomic DNA, while apoptotic cells cannot reverse nuclear fragmentation. We found a significant decrease in the tail moments of nuclei from mutagen-treated cells after 4 hr of liquid holding. However, this measurement may represent only those cells capable of repair. Apoptotic cells may continue DNA fragmentation during the recovery time and this DNA may become so diffuse that the nuclei disappear after electrophoresis. To overcome this possible artifact, images of nuclei were captured before and after alkaline electrophoresis. Constellations of nuclei were located on SCGE slides by their coordinates on the microscope stage. We found that no nuclei were lost due to apoptotic nuclear fragmentation and DNA migration. Even the so-called "hedgehog" comet images with extreme DNA damage were not lost during liquid holding. These data support the conclusion that mutagen-induced DNA damage is the principal cause of the damage measured in the comet assay.

A multiplex PCR/LDR assay for simultaneous detection and identification of the NIAID category B bacterial food and water-borne pathogens.

Enteric pathogens that cause gastroenteritis remain a major global health concern. The goal of this study was to develop a multiplex PCR/ligation detection reaction (LDR) assay for the detection of all NIAID category B bacterial food and water-borne pathogens directly from stool specimens. To validate the PCR/LDR assay, clinical isolates of Campylobacter spp., Vibrio spp., Shigella spp., Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and diarrheagenic Escherichia coli were tested. The sensitivity and specificity of the assay were assessed using a large number of seeded culture-negative stool specimens and a smaller set of clinical specimens from Haiti. The overall sensitivity ranged from 91% to 100% (median 100%) depending on the species. For the majority of organisms, the sensitivity was 100%. The overall specificity based on initial testing ranged from 98% to 100% depending on the species. After additional testing of discordant samples, the lowest specificity was 99.4%. PCR/LDR detected additional category B agents (particularly diarrheagenic E. coli) in 11/40 specimens from Haiti that were culture-positive for V. cholerae and in approximately 1% of routine culture-negative stool specimens from a hospital in New York. This study demonstrated the ability of the PCR/LDR assay to detect a large comprehensive panel of category B enteric bacterial pathogens as well as mixed infections. This type of assay has the potential to provide earlier warnings of possible public health threats and more accurate surveillance of food and water-borne pathogens.