We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.
Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.
Tilapia lake virus disease (TiLVD) is a viral disease in tilapia (Oreochromis spp.) that reportedly causes tilapia deaths in many regions of the world, including Asia1,2, Africa, and America. The disease was first recognized during the mass mortality of tilapia in 2009 in Israel, where the number of wild tilapia in Lake Kinneret plummeted dramatically from 257 to 8 tons per year2. The disease is caused by the tilapia lake virus (TiLV), which has been assigned to the family Amnoonviridae as a new genus Tilapinevirus and a new species Tilapia tilapinevirus3. Genetic characterization of TiLV showed that the virus is a novel enveloped, negative-sense, single-stranded RNA virus that has 10 segments encoding 10 proteins1,2,4. Various species of tilapia in the genus Sarotherodon, Oreochromis, and Tilapine and other warm water fish (e.g., giant gourami (Osphronemus goramy)) have been shown to be susceptible to TiLV2,5. Currently, this virus continues to spread globally, possibly through the movement of infected live fish6,7, while the risk of viral transmission via frozen tilapia or its product is limited8. Substantial mortality due to TiLV infection has the potential to have a significantly detrimental economic impact on the tilapia industry. For example, the economic impact of summer mortality syndrome in Egypt associated with TiLV infection was calculated to be US$100 million9. Accordingly, it is important to develop a rapid and proper diagnostic method to facilitate the control of this disease in fish farms.
Until now, the diagnosis of TiLVD has been based on molecular assays, viral isolation, and histopathology. Different PCR protocols and primers have been developed for TiLV diagnosis10,11. For instance, a SYBR green-based reverse transcription quantitative PCR (RT-qPCR) method with the sensitivity to detect as few as two copies/µL of the virus has been developed and validated for TiLV detection10. Other PCR methods for TiLV detection include TaqMan quantitative PCR11, RT-PCR2, nested RT-PCR12, and semi-nested RT-PCR13. However, these methods require sophisticated laboratory equipment and relatively extended periods of time to yield results due to the complexity of the reactions, which makes them unsuitable for field application.
The loop-mediated isothermal amplification (LAMP) assay is a rapid, simple, and practical for-field application14,15. The technique employs the principle of a strand displacement reaction, while the amplification reaction runs under isothermal conditions without a sophisticated and expensive thermal cycler14,15. Consequently, amplified LAMP products or RT-LAMP products are analyzed in ladder-like bands using agarose gel electrophoresis with a fluorescent stain for either the safe visualization of DNA or RNA14 or observation with the naked eye for the presence of turbidity or a white precipitate16,17,18. For these reasons, this technique has been used for the on-site detection of different fish pathogens17,18,19,20,21,22,23,24,25,26,27. The purpose of this study was to establish a rapid, sensitive, and accurate RT-LAMP assay for TiLV detection. The RT-LAMP assay offers screening for TiLV in fish samples within 30 min. The technique may be applied for the diagnosis and surveillance of TiLVD.
This experiment, which involved the use of animal tissue, was approved by the Institutional Animal Care and Use Committee of Kasetsart University, Bangkok, Thailand (protocol number ACKU61-VET-009).
1. Tissue collection
2. RNA extraction
3. Primer design
4. RT-LAMP assay
5. Agarose gel electrophoresis
6. Complementary DNA (cDNA) synthesis
7. RT-qPCR
In this study, an RT-LAMP assay was developed to detect TiLV infection in tilapia. The tested samples were collected from 14 farms located in different parts of Thailand between 2015 and 2016. The infected and uninfected fish were primarily grouped based on physical diagnoses and the appearances of symptomatic TiLVD. TiLV infection was subsequently confirmed using RT-PCR after the collection process. Agarose gel electrophoresis and the detection of a luminescent green color were selected as the evaluation methods of the LAMP amplicons (Figure 2). The liver and mucus of the infected and uninfected tilapia fish were characterized by the clinical appearance of TiLV disease symptoms, including skin erosion, skin redness, exophthalmos, and abdominal swelling. Previous reports have demonstrated the use of liver samples in molecular diagnostic assays to determine the presence of TiLV35. Alternatively, mucus may be beneficial in the assay as it may help avoid killing the animals. The results showed a ladder-like DNA band pattern and a fluorescent green color in the infected liver and mucus samples of infected fish (Figure 2), while no DNA band and the yellow color of calcein were observed in the RT-LAMP mixtures in uninfected animal tissues (Figure 2). Interestingly, a TiLV-infected tilapia sample collected from a farm in Malaysia was also diagnosed as positive using this RT-LAMP assay; however, variations in the PCR product’s size compared to the samples collected from local Thai farms were observed (Figure 2).
To verify the sensitivity and specificity of the RT-LAMP assay, a total of 63 TiLV-infected tissues and 63 uninfected tissues were analyzed using both the RT-LAMP and RT-qPCR assays (Table 1). A comparison of the RT-qPCR and RT-LAMP assays of the infected samples revealed a positive result in 63 (100%) and 51 (80.95%) of the samples, respectively. Moreover, an analysis of the uninfected samples showed that all 63 uninfected tissues yielded negative results using both the RT-qPCR and RT-LAMP assays (Table 1). This analysis demonstrated the reliability of the RT-LAMP assay for primary TiLV detection. To test the specificity of the RT-LAMP assay, tissue from fish infected with other pathogenic bacteria and viruses, including Streptococcus agalactiae, Francisella noatunensis, Flavobacterium columnare, Aeromonas hydrophila, and Iridovirus were used as templates for the RT-LAMP and RT-qPCR analyses. Both the RT-LAMP and RT-qPCR primers yielded negative results with no colorimetric changes and no fluorescent signals in any of the tested samples (Table 2). Additionally, the sensitivity of the RT-LAMP assay was assessed using a serial 10-fold dilution of the RNA templates extracted from TiLV-infected fish. Comparatively, the RT-qPCR assay was more sensitive than the RT-LAMP assay as the RT-qPCR assay had a detection limit of 10-8 while the RT-LAMP method required a 10-7-fold dilution to detect the TiLV genome (Table 2).
Figure 1. The nucleotide sequences of the six RT-LAMP primers used in this study that were specific to the detection of TiLV. The position of each primer was aligned on segment 3 of the TiLV genome (accession number KX631923). Please click here to view a larger version of this figure.
Figure 2. Analysis of the RT-LAMP amplicons obtained from the TiLV-infected and uninfected samples (A) in 1.5% agarose gel electrophoresis and (B) by fluorescent visualization. M = 1 kb DNA ladder, 1–2 = RNA from the livers of TiLV-infected fish, 3–4 = the cDNA of TiLV-infected fish, 5–6 = RNA from the mucus of TiLV-infected fish, 7–8 = cell lines of TiLV-infected fish, 9 = RNA from the livers of TiLV-infected fish (Malaysia), 10 = RNA from the livers of non-TiLV-infected fish, 11 = the cDNA of non-TiLV-infected fish, 12 = RNA from the mucus of non-TiLV-infected fish, 13 = no template control Please click here to view a larger version of this figure.
Types of samples | Numbers of samples | Detection validity (%) | |
RT-qPCR | RT-LAMP | ||
Infected samples | 63 | 100.00 (63/63) | 80.95 (51/63) |
Uninfected samples | 63 | 0 (0/63) | 0 (0/63) |
Table 1. Verification of RT-LAMP for TiLV detection in infected and uninfected fish samples using RT-qPCR and RT-LAMP
Specificity | S.a. | F.n. | F.c. | A.h. | I.v. | |||||
qPCR | LAMP | qPCR | LAMP | qPCR | LAMP | qPCR | LAMP | qPCR | LAMP | |
– | – | – | – | – | – | – | – | – | – | |
Sensitivity | method/ | 10-1 | 10-2 | 10-3 | 10-4 | 10-5 | 10-6 | 10-7 | 10-8 | 10-9 |
dilution | ||||||||||
qPCR | + | + | + | + | + | + | + | + | – | |
LAMP | + | + | + | + | + | + | + | – | – |
Table 2. Specificity and sensitivity of the RT-LAMP assay compared with RT-qPCR. For the specificity evaluation, RNA obtained from fish tissue infected with other bacteria or viruses, including Streptococcus agalactiae (S.a.), Francisella noatunensis (F.n.), Flavobacterium columnare (F.c.), Aeromonas hydrophila (A.h.), and Iridovirus (I.v.), was used as templates for RT-qPCR and RT-LAMP. For the sensitivity evaluation, RNA obtained from TiLV-infected fish was 10-fold serially diluted from 100 ng to 1 fg and used as templates for RT-qPCR and RT-LAMP. The + and – signs mean positive and negative results, respectively.
The aquaculture industry is continuously threatened by viral infections that cause substantial economic losses9,23,28. For instance, the emerging TiLV poses a major threat to tilapia-producing countries in many parts of the world1,6,9. Until now, there have been no specific therapeutics available to prevent TiLVD. While the development of a vaccine is ongoing, an efficient vaccine will require substantial time before it is available for commercial purposes. Given these circumstances, strict biosecurity measurements, such as the application of disinfectants, are necessary in fish farms to prevent the spread of TiLVD29,30. Currently, one of the most efficient control measures to reduce TiLV transmission is the screening of juvenile fish and adults for the presence or absence of TiLV31. For screening purposes, the diagnostic tool needs to be rapid, sensitive, and specific so that it can assist in eliminating infected populations and prevent the further spread of disease. However, the current molecular assays for TiLV are difficult to implement on-site. In the first instance, they require skillful researchers and expensive equipment. Second, it may take several days to obtain the laboratory results, which makes it difficult to control and prevent the spread of disease promptly15,16.
To overcome these problems, Notomi et al.14 established a novel nucleic acid-based amplification assay called loop-mediated isothermal amplification (LAMP) in 2000. The LAMP reaction has since been successfully applied to detect various fish viruses16,17,18,19,20,21,22,23,24,25,26,32,33,34. In this study, we developed an RT-LAMP protocol to detect TiLV in tilapia fish samples. Although the sensitivity of the RT-LAMP method is 10 times less efficient than that of the RT-qPCR, the RT-LAMP assay can detect the presence of a TiLV RNA genome as low as 100 fg, which is sufficient for TiLV detection in clinically diseased fish34. Notably, the RT-LAMP assay yields a result within 60 min and requires only a simple water bath or heat block instruments34, while the RT-qPCR assay takes more time and requires more expensive real-time PCR equipment for the analysis15,34. Moreover, the end product of the RT-LAMP was observed through a change in color of the fluorescent dye from light yellow to fluorescent green, making it visible to the naked eye without any requirement for sophisticated equipment34. These advantages make RT-LAMP suitable for field diagnosis. Furthermore, the study findings suggested that both liver and mucus can be used for TiLV detection using RT-LAMP. Similar to previous studies, a nonlethal sample using mucus allowed the diagnosis of TiLV without killing fish or valuable broodstock35. Recently, an RT-LAMP assay was developed to detect Chinese and Thai isolates of TiLV nucleotide sequences in segment 1 (S1 region) using a set of six primers36. The present study demonstrated that the developed RT-LAMP assay diagnosed a false-negative signal of TiLV infection at 27.78% compared to RT-PCR when using the same primer set. On further comparison, the false-negative result was relatively less at 19.05% when detecting segment 3 of TiLV compared to RT-PCR. When comparing the efficiency of the viral detection method with other reports, we were able to detect TiLV infection at 80.95%, while other works detected the virus at 72.22%36 and 82.89%37. Altogether, it may be hypothesized that the different components of the RT-LAMP assay, for example, the different primer sets and the different targeted segments of the TiLV genome are important factors influencing the validity of the assay.
Although the RT-LAMP assay is a powerful tool for disease screening and has several demonstrable benefits, this study was not without limitations. One of the critical points for the RT-LAMP assay was the design of an appropriate primer set comprising four to six primers. To promote the formation of the stem-loop structures of the PCR products, the appropriate lengths of the targeted genes or nucleotide sequences needed to be longer than about 500 bp, while the targeted genes of the RT-PCR assay had to be relatively short, in the range of 50–150 bp14,38,39.
In conclusion, the developed RT-LAMP assay is rapid, cost-effective, sensitive, and specific for TiLV detection. The analysis can be completed within 1 h compared with 4–5 h for the RT-qPCR assay. Notably, the RT-LAMP assay is practical for field conditions as positive results can be observed with the naked eye without requiring the use of sophisticated equipment.
The authors have nothing to disclose.
The project is financially funded by Thailand Research Fund (TRF) grant number RDG6050078 and the Center for Advanced Studies for Agriculture and Food, Institute for Advanced Studies, Kasetsart University, Bangkok, Thailand under the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, Ministry of Education, Thailand. The research is supported in part by the Graduate Program Scholarship from the Graduate School, Kasetsart University. The authors would like to thank Dr. Kwanrawee Sirikanchana for the narrative speaking of the video and Piyawatchara Sikarin for editing the video.
Tissue collection: | |||
Clove oil | Better Pharma | N/A | |
Tricaine methanesulfonate | Sigma-Aldrich | E10521 | An alternative option to clove oil |
RNA extraction: | |||
Acid guanidinium-phenol based reagent (TRIzol reagent) | ThermoFisher Scientific Corp. | 15596026 | |
Acid guanidinium-phenol based reagent (GENEzol reagent) | Geneaid | GZR100 | |
Direct-zol RNA Kit: | Zymo Research | R2071 | |
– Direct-zol RNA PreWash | |||
– RNA Wash Buffer | |||
– DNase/RNase-free water | |||
– Zymo-spin IIICG columns | |||
– Collection Tubes | |||
RT-LAMP: | |||
1x SD II reaction buffer | Biotechrabbit | BR1101301 | |
Magnesium sulfate (MgSO4) | Sigma-Aldrich | 7487-88-9 | |
dNTP set | Bioline | BIO-39053 | |
Betaine | Sigma-Aldrich | B2629 | |
Calcein mixture | Merck | 1461-15-0 | |
Bst DNA polymerase | Biotechrabbit | BR1101301 | |
AMV reverse transcriptase | Promega | M510A | |
Nuclease-free water | Invitrogen | 10320995 | |
Elite dry bath incubator, single unit | Major Science | EL-01-220 | |
Gel electrophoresis: | |||
Agarose | Vivantis Technologies | PC0701-500G | |
Tris-borate-EDTA (TBE) buffer | Sigma-Aldrich | SRE0062 | |
Tris-acetic-EDTA (TAE) buffer: | |||
– Tris | Vivantis Technologies | PR0612-1KG | |
– Acetic acid (glacial), EMSURE | Merck Millipore | 1000632500 | |
– Disodium Ethylenediaminetetraacetate dihydrate (EDTA), Vetec | Sigma-Aldrich | V800170-500G | |
Neogreen | NeoScience Co., Ltd. | GR107 | |
DNA gel loading dye (6X) | ThermoFisher Scientific Corp. | R0611 | |
DNA ladder and markers | Vivantis Technologies | PC701-100G | |
Mini Ready Sub-Cell GT (Horizontal electrophoresis system) | Bio-Rad | 1704487 | |
PowerPac HC power supply | Bio-Rad | 1645052 | |
Gel Doc EZ System | Bio-Rad | 1708270 | |
UV sample tray | Bio-Rad | 1708271 | |
NαBI imager | Neogene Science | ||
cDNA synthesis: | |||
ReverTra Ace qPCR RT Kit | Toyobo | FSQ-101 | |
Viva cDNA Synthesis Kit | Vivantis Technologies | cDSK01 | An alternative option for cDNA synthesis |
NanoDrop2000 (microvolume spectrophotometer) | ThermoFisher Scientific Corp. | ND-2000 | |
T100 Thermal Cycler | Bio-Rad | 1861096 | |
RT-qPCR: | |||
iTaq Universal SYBR Green Supermix | Bio-Rad | 1725120 | |
Nuclease-free water, sterile water | MultiCell | 809-115-CL | |
8-tube PCR strips, white | Bio-Rad | TLS0851 | |
Flat PCR tube 8-cap strips, optical | Bio-Rad | TCS0803 | |
CFX96 Touch Thermal Cycler | Bio-Rad | 1855196 | |
General equipment and materials: | |||
Mayo scissors | N/A | ||
Forceps | N/A | ||
Vortex Genie 2 (vortex mixer) | Scientific Industries | ||
Microcentrifuge LM-60 | LioFuge | CM610 | |
Corning LSE mini microcentrifuge | Corning | 6765 | |
Pipettes | Rainin | Pipete-Lite XLS | |
QSP filtered pipette tips | Quality Scientific Plastics | TF series | |
Corning Isotip filtered tips | Merck | CLS series | |
Nuclease-free 1.5 mL microcentrifuge tubes, NEST | Wuxi NEST Biotechnology | 615601 |