Rapid and sensitive detection of infectious spleen and kidney necrosis virus by recombinase polymerase amplification combined with lateral flow dipsticks

Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards

“…If the corresponding genome copy numbers were deduced, that value of LOD in weight would correspond to 1.91 × 10 5 copies/mL (1.71 × 10 2 cps/reaction), which seems to be a little higher with respect to the results obtained for other RNA viruses such as VHSV and infectious hematopoietic necrosis virus (IHNV) [ 26 , 27 ]. However, in terms of pDNA and iv RNA copies, the LODs obtained with this procedure (2.36 and 7.86 copies/mL, respectively) were clearly below those reported for both RNA [ 26 , 28 , 29 , 30 , 31 ] and DNA [ 32 , 33 , 34 , 35 , 36 ] fish viruses. This discrepancy is related to the large difference observed between the LOD value as crude virus RNA copies and that of the rest of standards, which was over 4 Log 10 (from 1.17 × 10 4 to 8.09 × 10 4 times higher; data extracted from Table 2 ).…”

Rapid and sensitive detection of pathogenic Elizabethkingia miricola in black spotted frog by RPA-LFD and fluorescent probe-based RPA

“…The basic RPA was initiated by using the TwistAmp Basic kit (TwistDx, Cambridge, UK), and the reaction system contained 29.5 μL rehydration buffer, 2 μL forward primer, 2 μL reverse primer, 12 μL ddH 2 O and 2 μL E. miricola DNA sample, and then 2.5 μL magnesium acetate was added immediately to start the reaction, and the reaction lasted for 30 min at 38 °C [17] . H 2 O was used as a negative control.…”

“…For instance, RPA is used to detect genotype III grass carp reovirus [14] , shrimp hemocyte iridescent virus [15] and spring viremia of carp virus [16] . Previous reports described that RPA was more sensitive, rapid and accurate than traditional PCR [17] . Although the traditional RPA can achieve the detection of RPA products by agarose gel electrophoresis, the operation process is prone to cross-contamination and is not suitable for clinical detection [18] .…”

“…Recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) is an innovative detection method suitable for practical application. With the advantages of visual and handy, RPA-LFD has received extensive attention [ 17 , 19 , 20 ]. This method not only avoids the possibility of cross-contamination, but also eliminates the need for bulky laboratory equipment and cumbersome handling steps.…”

“…The results are visualized in less than 30 min. RPA-LFD is used to detect infectious spleen and kidney necrosis virus [17] , cyprinid herpesvirus 2 [20] , African swine fever virus [19] , goatpox virus and sheeppox virus [21] , Escherichia coli O157:H7 [22] and salmonella spp. in food samples [23] .…”