Loop-mediated isothermal amplification for the detection of goose circovirus

Abstract

BackgroundGoose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection.ResultsThe presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods.ConclusionsThe developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.

Loop-Mediated Isothermal Amplification as a Simple Molecular Method for the Detection of Derzsy’s Disease Virus

“…The explanation for these results is the mechanism of LAMP that requires the use of outer and inner primers thus following very sensitive nested-PCR. The third pair of loop primers is not necessary for LAMP progress but significantly accelerates the reaction (11)(12)(13).…”

“…The authors obtained positive results after 20 min at 65°C (13). The specificity of the reaction was tested using different pathogens (Aleutian disease virus-ADV, Newcastle disease virus-NDV, Pasteurella multocida, Salmonella Enteritidis, Escherichia coli), while the sensitivity was tested using serial 10-fold dilutions of plasmid DNA (pVP3 2.8 x10 0 -2.8x10 11 ). Similar to our data, the authors have demonstrated that LAMP is as specific as PCR (13).…”

“…LAMP mixture contains SYBR Green or Gel Red TM dye, which binds to the amplified DNA what results in a strong fluorescence in tubes containing target DNA. Moreover, LAMP does not need gel electrophoresis since the results may be seen by a naked eye or under UV light illumination (7)(8)11).…”

Increasing importance of genomics in recognition of infectious animal diseases taking into account OIE requirements

“…Omawiane techniki genetyczne, coraz częściej i szerzej zastępują testy opierające się na ocenie właściwościach fenotypowych drobnoustrojów i reakcjach antygen-przeciwciało (3,6,23,24). Dotychczas opracowano wiele technik wykrywania i analizy materiału genetycznego bakterii i wirusów, z których najbardziej istotnymi wydają się: łańcuchowa reakcja polimerazy (polymerase chain reaction -PCR) (25,26,36) wraz z jej pochodnymi modyfikacjami oraz techniki amplifikacji DNA w warunkach stałej temperatury (29,40).…”

Development of a rapid assay to detect the jellyfish Cyanea nozakii using a loop-mediated isothermal amplification method

“…For example, the LAMP reaction does not require expensive laboratory equipment such as a thermal cycler; the result is obtained within 1 h or less; the result shows specificity because 4-6 primers are required that hybridize against 6-8 distinct sequences in the target DNA; and the amplified products are visualized simply through an increase in turbidity or change in color detected through the use of an intercalating fluorescent dye such as SYBR Green I (Mori et al, 2001;Notomi et al, 2000). LAMP-based assays have been used for rapid and sensitive detection of microorganisms and harmful microalgae (Chen & Cui, 2009;Chen et al, 2013;Nagai et al, 2012;Picon-Camacho et al, 2013;Woźniakowski et al, 2012;Zhang et al, 2013Zhang et al, , 2014. The aim of the present study was to develop a sensitive, specific, and cost-effective LAMP-based approach for the detection of the harmful jellyfish C. nozakii.…”