An RT-PCR panel for rapid serotyping of dengue virus serotypes 1 to 4 in human serum and mosquito on a field-deployable PCR system.

BACKGROUND: Dengue fever, a mosquito-borne disease, is caused by dengue virus (DENV) which includes four major serotypes (DENV-1, -2, -3, and -4). Some serotypes cause more severe diseases than the other; severe dengue is associated with secondary infections by a different serotype. Timely serotyping can provide early warning of dengue epidemics to improve management of patients and outbreaks. A mobile insulated isothermal PCR (iiPCR) system is available to allow molecular detection of pathogens near points of need. METHODOLOGY/PRINCIPLE FINDINGS: In this study, side-by-side comparison with the CDC DENV-1-4 Real Time RT-PCR (qRT-PCR) was performed to evaluate the performance of four singleplex DENV-1-4 serotyping reverse transcription-iiPCR (RT-iiPCR) reagents for DENV subtyping on the mobile PCR system. The four RT-iiPCRs did not react with Zika virus and chikungunya virus; tests with serial dilutions of the four DENV serotypes made in human serum showed they had detection endpoints comparable to those of the reference method, indicating great analytical sensitivity and specificity. Clinical performance of the RT-iiPCR reagents was evaluated by testing 40 serum samples each (around 20 target serotype-positive and 20 DENV-negative); all four reagents had high agreement (97.5-100%) with the reference qRT-PCR. Moreover, testing of mosquitoes separately infected experimentally with each serotype showed that the four reagents detected specifically their target DENV serotypes in mosquito. CONCLUSIONS/SIGNIFICANCE: With analytical and clinical performance comparable to the reference qRT-PCR assay, the four index RT-iiPCR reagents on the field-deployable PCR system can serve as a useful tool for DENV detection near points of needs.

Rapid and sensitive insulated isothermal PCR for point-of-need feline leukaemia virus detection.

Objectives Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection.

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

BACKGROUND: Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. RESULTS: Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. CONCLUSIONS: The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

Factors influencing the intention to utilize out-of-pocket health checkup services: A sample of citizens from 12 townships of Taichung County in Taiwan.

BACKGROUND: Taiwan started its National Health Insurance (NHI) system in 1995. However, until now, most cancer screening tests and preventive care have been out-of-pocket (OOP) medical items excluded from the coverage of NHI. The aim of this study was to explore the factors influencing an individual's intention to utilize OOP health checkups. METHODS: A cross-sectional research method was adopted in this study. Based on the theory of planned behavior, a questionnaire was developed and used to survey purposively sampled residents (n = 940) from 12 randomly selected townships in Taichung County, Taiwan, from August to September 2006. Descriptive statics and linear regression were conducted to analyze the collected data. RESULTS: Our results showed that result evaluation (beta = 0.092), behavioral beliefs (beta = 0.088), behavioral norms of people with experience in utilizing OOP health checkups (beta = 0.116), perceived convenience (beta = 0.273), and worry about illness and perceived health (beta = 0.110) were important factors influencing the intention to utilize OOP health checkups. Age, education and acceptable health checkup charges were also related. CONCLUSION: Reinforcing disease- and health checkup-related knowledge may positively influence an individual's intention to utilize OOP health checkups. In addition, improving perceived convenience and reducing disease-screening barriers can intensify the individual's intention to use OOP health checkups. The influence of age, education level and OOP checkup charges should also be taken into consideration when related policies are formulated.

Specific detection of reverse transcription-loop-mediated isothermal amplification amplicons for Taura syndrome virus by colorimetric dot-blot hybridization.

The goal of this study was to develop a field diagnosis system based on isothermal reverse transcription-loop-mediated amplification (RT-LAMP) for shrimp Taura syndrome virus (TSV), placing emphasis on specific and simple detection of the LAMP amplicons. After a single-tube RT-LAMP reaction for TSV was established, colorimetric dot-blot hybridization (DBH) was adopted to detect signals only from the target-derived amplicons. The results showed that the modified DBH offered unambiguous and sensitive detection of the TSV RT-LAMP amplicons without the UV cross-linking and denaturation steps. Together, TSV RT-LAMP-DBH assay reached the same dilution point as reverse transcription-nested polymerase chain reaction-agarose gel electrophoresis (RT-nPCR-AGE) for TSV detection. Specificity of the assay was demonstrated by the absence of DBH signal from yeast tRNA and various shrimp viruses. TSV RT-LAMP-DBH was applied to 125 Penaeus vannamei and demonstrated a very good concordance (kappa value, 0.823) with RT-nPCR-AGE assay in detection efficiency. Furthermore, a one-step guanidinium thiocyanate (GuSCN) homogenization method was established to provide RNA extraction efficiency comparable to that of the TRIzol Reagent for RT-LAMP. Requiring simply a heating apparatus, the GuSCN RNA extraction-isothermal RT-LAMP-DBH protocol has the potential for further development for diagnosis of diseases in the field.

Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei by ramification amplification assay.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus that causes developmental and growth abnormalities in Pacific white shrimp Litopenaeus vannamei (also known as Penaeus vannamei). Nucleic acid based methods such as in situ hybridization (ISH) and PCR have been commonly used for IHHNV detection. Ramification amplification (RAM), an isothermal nucleic acid amplification approach, was used in this study to detect IHHNV in L. vannamei. RAM offers many advantages over PCR, including simple procedures and short detection time, and is labor-saving and cost-effective. RAM exponentially amplifies a circular oligonucleotide amplicon (C probe) after a target-specific ligation step through sequential primer extension and strand displacement processes. The conditions of an IHHNV RAM assay were optimized using artificial templates and targets prior to application. Using DNA of IHHNV-infected L. vannamei as targets, results revealed that RAM amplified target DNA with similar sensitivity as PCR. RAM offers competitive levels of speed, simplicity and sensitivity among various pathogen diagnostic methods.