Colorimetric Method of Loop‐Mediated Isothermal Amplification with the Pre‐Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms

Abstract

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.

Detection of natural infection of infectious spleen and kidney necrosis virus in farmed tilapia by hydroxynapthol blue-loop-mediated isothermal amplification assay

“…Therefore, LAMP assay is indeed a sensitive tool for identifying covert infections in fish and can be a valuable technique for screening pathogens in aquaculture in general (Suebsing et al . , ).…”

Vertical transmission and concurrent infection of multiple bacterial pathogens in naturally infected red tilapia (Oreochromisspp.)

“…; Suebsing, Kampeera, Sirithammajak, Withyachumnarnkul et al . ; Suebsing, Kampeera, Sirithammajak, Pradeep et al . ) and is therefore the most appropriate means for rapidly and reliably testing tilapia for the presence of pathogens.…”

“…Total DNA was extracted from each individual sample and from 5 ml of bacteria culture from reference and farm isolates (Table ) using a rapid extraction solution (0.025 N sodium hydroxide and 0.0125% sodium dodecyl sulphate) as previously reported (Suebsing, Kampeera, Sirithammajak, Withyachumnarnkul et al . ). Extracted nucleic acids were quantified using nanodrop (Thermo Scientific, Waltham, MA, USA) and was diluted to a final concentration of 100 ng μL −1 , and 2 μL of the resulting solution was determined for cytochrome oxidase I gene (COI) (Kowasupat, Panijpan, Laosinchai, Ruenwongsa, Phongdara, Wanna, Senapin & Phiwsaiya ) amplification before used as the template for the LAMP assay.…”

“…So far, disease studies on tilapia have overlooked the synergistic effect of mixed pathogens and the risk of vertical transmission of these pathogens. Recent studies have provided some evidence that vertical transmission could be a route for transmission of some diseases between generations of tilapia (Suebsing, Kampeera, Tookdee, Withyachumnarnkul, Turner & Kiatpathomchai ; Suebsing, Kampeera, Sirithammajak, Pradeep, Jitrakorn, Arunrut, Sangsuriya, Saksmerprome, Senapin, Withyachumnarnkul & Kiatpathomchai ; Suebsing, Kampeera, Sirithammajak, Withyachumnarnkul, Turner & Kiatpathomchai ; Pradeep, Suebsing, Sirthammajak, Kampeera, Jitrakorn, Saksmerprome, Turner, Palang, Vanichviriyakit, Senapin, Kiatpathomchai & Withyachumanarnkul ). However, confirmation of vertical transmission for other commonly reported pathogens in tilapia farming is still lacking.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…Positive LAMP reaction was indicated with green colour, whereas negative LAMP reaction indicated with orange colour. To name a few, calcein has been used in detection of Trypanosoma cruzi (Besuschio et al 2017), avian influenza H5 virus (Liu et al 2013), hepatitis C virus (Zhao et al 2017), human papillomavirus , human coronavirus (Geng et al 2016) and Flavobacterium columnare in tilapia fish (Suebsing et al 2015). Calcein-based end-point detection is most commonly used due to its high sensitivity and notable colour change for naked eye observation (Fischbach et al 2015).…”