These results demonstrate that our HBV-LAMP assay is rapid, sensitive and specific, and capable of detecting the major HBV genotypes. This assay could be used in clinical point-of-care settings, mainly in endemic and resource-limited environments for HBV diagnostics, donor screening, epidemiological studies, and therapeutic monitoring of patients undergoing antiviral treatment.

These diagnostic characteristics highlight the potential utility of this assay for the rapid detection and genotype identification of HCV infection in field and point-of-care settings in endemic regions and resource-limited environments.

A rapid multiplex isothermal amplification assay has been developed for detection and identification of multiple blood-borne viruses that infect millions of people world-wide. These infections may lead to chronic diseases or death if not diagnosed and treated in a timely manner. Sets of virus-specific oligonucleotides and oligofluorophores were designed and used in a reverse-transcription loop-mediated multiplexed isothermal amplification reaction for detection and gel electrophoretic identification of human Immunodeficiency virus (HIV), hepatitis-B virus (HBV), hepatitis-C virus (HCV), hepatitis-E virus (HEV), dengue virus (DENV), and West Nile (WNV) virus infection in blood plasma. Amplification was catalyzed with two thermostable enzymes for 30–60 minutes under isothermal condition, utilizing a simple digital heat source. Electrophoretic analysis of amplified products demonstrated simultaneous detection of 6 viruses that were distinctly identified by unique ladder-like banding patterns. Naked-eye fluorescent visualization of amplicons revealed intensely fluorescing products that indicated positive detection. The test demonstrated a 97% sensitivity and a 100% specificity, with no cross-reaction with other viruses observed. This portable detection tool may have clinical and field utility in the developing and developed world settings. This may enable rapid diagnosis and identification of viruses for targeted therapeutic intervention and prevention of disease transmission.

Background: Traditionally, the detection and identification of Vibrio cholerae, V. parahaemolyticus and V. vulnificus has depended on thiosulfate citrate bile salt sucrose (TCBS) agar or CHROMagar Vibrio and then identified with biochemical tests as a gold standard. However, this process limits their performance efficiency and rapidity and hence simultaneous detection of the responsible organisms in laboratory settings is required.Methods: A novel sensitive, locus-specific single tube multiplex polymerase chain reaction (STMPCR) is developed to amplify genetically stable target DNA sequences of V. cholerae (ompW), V. parahaemolyticus (tl), or V. vulnificus (vvhA), and subsequently, the amplifications of these discrete loci yield amplicons with expected sizes at 588, 450, and 383 bp, respectively.Results: The STMPCR have very high specificity, these primers give correctly the amplification with gDNAs from 163 isolates of V. cholerae, 233 isolates of V. parahaemolyticus and 59 isolates of V. vulnificus which are obtained from food and water samples. The false positives in the reaction containing gDNA of 94 isolates from Vibrio spp. and other bacteria are not observed. In addition, the analytical sensitivity of STMPCR is 1 pg per reaction when amplify single gDNA or 10 pg per reaction when amplify both of mixed two and mixed three gDNAs. Furthermore, the analytical sensitivity of Vibrio suspension in normal saline and in homogenate shrimp sample when amplify single, mixed two, and mixed three Vibrio are 10 cfu, 102 cfu and 103 cfu per reaction, respectively. The STMPCR technique is applied for detecting these three Vibrio spp. in 30 shrimp samples, and the results show that V. parahaemolyticus, V. cholerae-V. parahaemolyticus, and V. parahaemolyticus-V. vulnificus are detected in 67%, 6% and 27%, respectively. Conclusion:As a result, STMPCR can be used as an alternative method for direct detection and/or identification of V. cholerae,V.parahaemolyticus and V. vulnificus isolates in environment samples.