Rapid and sensitive detection of Senecavirus A by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick method

Abstract

Senecavirus A (SVA) is a critical pathogen causing vesicular lesions in sows and acute death of newborn piglets, resulting in very large economic losses in the pig industry. To restrict the transmission of SVA, an establishment of an effective diagnostic method is crucial for the prevention and control of the disease. However, traditional detection methods often have many drawbacks. In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) was combined with a lateral flow dipstick (LFD) to detect SVA. The resulting RT-LAMP-LFD assay was performed at 60°C for 50 min and then directly judged on an LFD visualization strip. This method shows high specificity and sensitivity to SVA. The detection limit of RT-LAMP was 4.56x10 -8 ng/μL RNA, approximately 11 copies/μL RNA, and it was 10 times more sensitive than RT-PCR. This detection method’s positive rate for clinical samples is comparable to that of RT-PCR. This method is time saving and highly efficient and is thus expected to be used to diagnose SVA infections in this field.

Fast and User-Friendly Detection of Flatfish Species (Pleuronectes platessa and Solea solea) via Loop-Mediated Isothermal Amplification (LAMP)

“…Since no LF primer was available, we used labeled FIP and BIP primers with 5′-biotin and 5′-FAM, respectively. This combination has been used several times and is characterized by high specificity and the ability to be used for multiplexing. − Figure A shows the assay without an added loop primer. The LAMP reaction was incubated at 72 °C for 15 min.…”

Rapid and Visual Detection of Vibrio parahaemolyticus in Aquatic Foods Using blaCARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD)

“…To our knowledge, the present study is the first report of LAMP and LAMP-LFD assays specifically based on the bla CARB-17 gene for detecting V. parahaemolyticus in aquatic foods. The reaction conditions and systems of LAMP-LFD, such as Mg 2+ concentration, primer concentration, and enzyme concentration, can directly affect the sensitivity and specificity of the detection [ 35 ]. In our study, the optimization of LAMP-LFD conditions was first comparatively achieved under different concentrations of Mg 2+ and dNTP, concentration ratios of the inner primer to the outer primer, Bst DNA polymerase quantity, and reaction temperature and time, since these parameters may influence the amplification efficiency of the LAMP-LFD assay [ 10 ].…”

Advances in the differential molecular diagnosis of vesicular disease pathogens in swine

“…Zeng et al described a real time RT-LAMP targeting the VP1 and VP2 regions of SVA and this assay can detect at least 1 TCID 50 /mL of virus titers ( Zeng et al, 2018 ). Li et al combined RT-LAMP with a lateral flow dipstick (LFD) for spot rapid diagnosis of SVA ( Li et al, 2019 ). Armson et al established SVV-1 RT-LAMP assay using lyophilized reagents, which negated the requirement for RNA extraction and the time of sample amplification was reduced within 12 min ( Armson et al, 2019b ).…”