Development and clinical evaluation of a rapid diagnostic kit for feline leukemia virus infection

Feline leukemia virus (FeLV) causes a range of neoplastic and degenerative diseases in cats. To obtain a more sensitive and convenient diagnosis of the disease, we prepared monoclonal antibodies specific for the FeLV p27 to develop a rapid diagnostic test with enhanced sensitivity and specificity. Among these antibodies, we identified two clones (hybridomas 8F8B5 and 8G7D1) that specifically bound to FeLV and were very suitable for a diagnostic kit. The affinity constants for 8F8B5 and 8G7D1 were 0.35 x 10(9) and 0.86 x 10(9), respectively. To investigate the diagnostic abilities of the rapid kit using these antibodies, we performed several clinical studies. Assessment of analytical sensitivity revealed that the detection threshold of the rapid diagnostic test was 2 ng/mL for recombinant p27 and 12.5 x 10(4) IU/mL for FeLV. When evaluating 252 cat sera samples, the kit was found to have a kappa value of 0.88 compared to polymerase chain reaction (PCR), indicating a significant correlation between data from the rapid diagnostic test and PCR. Sensitivity and specificity of the kit were 95.2% (20/21) and 98.5% (257/261), respectively. Our results demonstrated that the rapid diagnostic test would be a suitable diagnostic tool for the rapid detection of FeLV infection in cats.

Surveillance of Acute Gastroenteritis in Seoul, Korea, During May 2004 and June 2007

Acute gastroenteritis (AGE), which is one of the most common diseases worldwide, primarily occurs in infants and young children in both developed and developing countries. To investigate the prevalence of AGE in Korea, 6,788 stool specimens collected from hospitalized patients with AGE in Seoul, Korea from March 2004 to June 2007 were analyzed by enzyme immunoassay, reverse transcription-PCR, DNA sequencing and phylogenetic analysis. Enteric viruses and bacteria were detected in 2,955 (43.5%) and 1,389 (20.5%) specimens, respectively. Among the enteric viruses detected, rotavirus (19.7%) and norovirus (18.9%) were the predominant causative agents, followed by adenovirus (2.5%) and astrovirus (2.4%). Staphylococcus aureus was the most commonly observed bacteria (8.0~19.2%). The epidemic peaks of the enteric viruses were October to December for norovirus, January to May for rotavirus, and August to October for adenovirus. The seasonal activity of rotavirus was shifted from winter to late spring. However, astrovirus did not display seasonal activity in this study. Although viral AGE primarily occurred in patients younger than 5 years of age, the incidence of viral AGE in children aged 6 to 14 years was significant. The results of this study will contribute to the currently available epidemiological data and improve public health and hygiene via amelioration of diagnostic methods and longitudinal surveillance.

Development of RT-PCR Method to Detect Various Human Enteric Viruses

Human enteric viruses are one of the major causes of acute gastroenteritis outbreaks. A rapid and precise detection of virus is critical for prompt diagnosis. For this purpose, nucleic acid-based techniques such as reverse transcription (RT)-PCR have been developed. Although RT-PCR is a rapid, specific and sensitive method to detect virus, many steps or reactions are required, especially when various types of viruses are targeted. In this study, we developed a quick and effective method to detect human enteric viruses with a few reactions. Our candidate viruses were as follows: one DNA virus (adenovirus: AdV) and seven RNA viruses including poliovirus (PV), coxsackievirus A (CoxA) and B (CoxB), human rotavirus (HRV), hepatitis A virus (HAV), norovirus (NorV), and astrovirus (AstV). With this amount of samples, theoretically, a total of fifteen biomolecular reactions have to be performed, which include seven RT reactions and eight subsequent PCR with specific primers in each case. Specific primers, enterovirus universal primers, and random primers were applied independently to compare the outcomes of RT and PCR steps in each viral sample. We found that random 9-mer is ideal for the RT reactions of RNA viruses with negligible differences in sensitivity and specificity of viral detection except HRV. Hence, HRV cDNA generated by HRV-specific primer and AdV DNA were amplified in a single tube by duplex PCR. The cDNAs generated by RT using random 9-mers were divided into two reaction tubes without losing sensitivity: one duplex PCR detects enteroviruses (PV, CoxA, CoxB) and HAV, the other detects NorV and AstV. In conclusion, it is possible to detect eight enteric viruses with a substantially reduced number of reactions, which are composed of five reactions, two RT and three PCR reactions.

Detection of Human Parvovirus B19 in Human Blood by Polymerase Chain Reaction

Viruses present in the blood or blood products serve important infection source to transfusion patients or users of blood products. Human parvovirus B19 has been recognized as a new viral pathogen in human mainly transmitted via blood. Thus, detection of human parvovirus B19 has become an urgent problem to be solved. This study was intended to develop methods to detect human parvovirus B19 in the blood or blood products by nucleic acid amplification technique (NAT) or polymerase chain reaction (PCR). Five sets of primer DNAs were tested for the detection of human parvovirus B19 by PCR. A primer set amplifying 258 nucleotides corresponding Vp1 gene of human parvovirus B19 was chosen and further studies were done to determine the optimum condition to detect human parvovirus B19 from human blood or blood products. PCR detection of human parvovirus B19 was almost 1,000 times more sensitive than the receptor-mediated hemagglutination assay developed by the Japanese Red Cross Center. Although direct PCR of B19 virus without DNA extraction could detect B19 virus from PBS buffer, attempts to detect the virus from whole blood or plasma failed. PCR after DNA extraction from blood or plasma samples could detect B19 virus as little as 104 PFU/ml. Our results can further be applied for developing routine methods to identify human parvovirus B19 in human blood or commercial blood products.

Effect of Cyclic GMP on Human Cytomegalovirus Gene Expression

No abstract available.