Real-time quantitative loop-mediated isothermal amplification as a simple method for detecting white spot syndrome virus

Abstract

Aims: White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one‐step, single tube, real‐time accelerated loop‐mediated isothermal amplification (real‐time LAMP) for quantitative detection of WSSV. Materials and Methods: A set of six specially designed primers that recognize eight distinct sequences of the target. The whole process can be completed in 1 h under isothermal conditions at 63°C. Detection and quantification can be achieved by real‐time monitoring in an inexpensive turbidimeter based on threshold time required for turbidity in the LAMP reaction. A standard curve was constructed by plotting viral titre against the threshold time (Tt) using plasmid standards with high correlation coefficient (R2 = 0·988). Conclusions: Sensitivity analysis using 10‐fold dilutions (equivalent to 35 ng μl−1 to 35 ag μl−1) of plasmid standards revealed this method is capable of detecting upto 100 copies of template DNA. Cross‐reactivity analysis with DNA/cDNA of IHHNV, TSV, YHV‐infected and healthy shrimp showed this method is highly specific for quantitative detection of WSSV. Significance and Impact of the Study: WSSV real‐time LAMP assay appears to be precise, accurate and a valuable tool for the detection and quantification of WSSV in large field samples and epidemiological studies.

Development and validation of a quantitative real-time polymerase chain assay for universal detection of the White Spot Syndrome Virus in marine crustaceans

“…The upper and lower quantification limits were 1.24 × 10 7 and 12 copies per PCR reaction, respectively. This detection limit is as low as that reported by using a nested PCR protocol [32], a fluorescent quantitative PCR protocol [52] or a loop-mediated isothermal amplification method [21], and more sensitive than the nested PCR protocol previously reported [53] (which may require a minimum of 1000 copies [54]).…”

Rapid Detection of Shrimp White Spot Syndrome Virus by Real Time, Isothermal Recombinase Polymerase Amplification Assay

“…Most of the methods for detecting WSSV target vp19 [35], vp28 [22], [26], [28], wsv360 [30], ORF191 [32], [33] and ORF 36 [36]. Among these genes, the vp28 is mostly widely used.…”

Validation of a Commercial Insulated Isothermal PCR-based POCKIT Test for Rapid and Easy Detection of White Spot Syndrome Virus Infection in Litopenaeus vannamei

“…Recently, various loop-mediated isothermal amplification (LAMP) assays have been developed for WSSV detection [23], [24], [25], [26], [27]. Requiring a simple incubator, LAMP also has the potential to be applied to point-of-need detection of aquaculture pathogens [28], [29].…”