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BackgroundDengue 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 findingsIn 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/SignificanceWith 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.

Background: Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKIT TM 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.

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. KEY WORDS: IHHNV · Litopenaeus vannamei · Ramification amplification · Isothermal amplification Resale or republication not permitted without written consent of the publisherDis Aquat Org 73: [103][104][105][106][107][108][109][110][111] 2006 Ramification amplification (RAM), a novel DNA amplification method, was first described in 1998 by Zhang et al. (1998). RAM is an isothermal amplification method that utilizes a circularizable oligodeoxyribonucleotide probe (C probe) as an amplifiable target. The C probe is a synthetic oligonucleotide with a sequence complementary to the specific target sequence. Upon hybridization to the target DNA, the 5' and 3' ends of the C probe are drawn together and can be ligated to form a closed circular molecule (Nilsson et al. 1994). Under isothermal condition, the circular C probe is subsequently amplified with a pair of C probe-specific primers, driven by Bst DNA polymerase with high strand-displacement activity. This amplification is also named hyperbranched rolling circle amplification or cascade rolling circle amplification.After RAM was first demonstrated by Zhang et al. (1998), it became widely applied to DNA-, RNA-or protein-based diagnosis and single nucleotide polymorphism analysis (Barany 1991, Tyagi et al. 1996, Lizardi et al. 1998, Thomas et al. 1999. Other isothermal amplification methods, such as loop-mediated isothermal amplification (LAMP), have also been reported to detect targets at the fentogram range under isothermal conditions (Kono et al. 2004). However, RAM includes a ligation step that offers better specificity over other isothermal amplification strategies (Zhang et al. 1998). If mismatch occurs at the ends of the C probes at the annealing stage, the ligation step in RAM would not proceed to allow the subsequent signal amplification step. Furthermore, RAM could yield more than 10 6 copies of amplicons within 1 h fr...

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