- Email: [email protected]
Rapid detection of tilapia lake virus using a one-step reverse transcription loop-mediated isothermal amplification assay
Accepted Manuscript Rapid detection of tilapia lake virus using a one-step reverse transcription loop-mediated isothermal amplification assay
Theerawut Phusantisampan, Puntanat Tattiyapong, Palita Mutrakulcharoen, Malinee Sriariyanun, Win Surachetpong PII: DOI: Reference:
S0044-8486(18)32808-4 https://doi.org/10.1016/j.aquaculture.2019.04.015 AQUA 634049
To appear in:
aquaculture
Received date: Revised date: Accepted date:
24 December 2018 21 February 2019 3 April 2019
Please cite this article as: T. Phusantisampan, P. Tattiyapong, P. Mutrakulcharoen, et al., Rapid detection of tilapia lake virus using a one-step reverse transcription loopmediated isothermal amplification assay, aquaculture, https://doi.org/10.1016/ j.aquaculture.2019.04.015
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ACCEPTED MANUSCRIPT Rapid detection of tilapia lake virus using a one-step reverse transcription loopmediated isothermal amplification assay Theerawut Phusantisampan1 , Puntanat Tattiyapong2,3 , Sriariyanun4 , Win Surachetpong2,3 *
Palita Mutrakulcharoen4 ,
Malinee
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Department of Biotechnology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok (KMUTNB), Thailand. 2
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Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Thailand. 3
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Center for Advanced Studies for Agriculture and Food, Kasetsart University, Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand (CASAF, NRU-KU), Thailand.
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The Sirindhorn Thai-German International Graduate School of Engineering (TGGS), King Mongkut's University of Technology North Bangkok (KMUTNB), Thailand.
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Abstract
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Tilapia lake virus (TiLV) is a newly emerging viral disease in tilapia with recent outbreaks in many parts of the world. There is an urgent need to develop an accurate, sensitive, on-farm method for rapid detection of TiLV to limit economic loss and prevent the
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spread of the virus into new geographical areas. In this study, we developed a rapid, one-step reverse transcription, loop-mediated, isothermal amplification (RT-LAMP) method for the
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detection of TiLV in fish tissues. Our results revealed that the RT-LAMP assay was able to detect TiLV infection in infected cell culture materials, and fish samples collected from different geographic locations in Thailand. In total, 166 tissue samples were collected from TiLV-infected fish and uninfected fish from tilapia farms in Thailand with a history of TiLV
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outbreaks, and fish showing clinical signs of TiLV infection were tested using our RT-LAMP protocol. The RT-LAMP assay offered high specificity and sensitivity compared to reverse transcription polymerase chain reaction (RT-qPCR) assay. These results supported the application of the RT-LAMP assay for the diagnosis of TiLV with benefits including reduced analysis time and easy interpretation of results based on the colorimetric change, thus offering a diagnostic tool in resource-limited countries where there is an urgent need for rapid diagnostic assay to guide TiLV control. Keywords: RT-LAMP, tilapia lake virus, diagnosis, tilapia Corresponding author: [email protected]
ACCEPTED MANUSCRIPT 1. Introduction Tilapia lake virus disease (TiLVD), caused by the tilapia lake virus (TiLV), is an emerging viral disease which has been recently reported in countries where tilapia is the main aquaculture species. The virus has been detected in wild and farm-raised tilapia and is associated with high mortality such as in summer mortality syndrome (Nicholson et al., 2017), one-month mortality syndrome (Surachetpong et al., 2017) and syncytial hepatitis
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disease (Del-Pozo et al., 2017). Currently, diagnosis of TiLV infection has relied on the detection of viral genomic RNA using reverse transcription polymerase chain reaction (RT-
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PCR) (Dong et al., 2017; Eyngor et al., 2014; Kembou Tsofack et al., 2017), RT-quantitative PCR (RT-qPCR) (Kembou Tsofack et al., 2017; Nicholson et al., 2018; Tattiyapong et al.,
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2018; Waiyamitra et al., 2018), virus isolation in the cell culture (Eyngor et al., 2014; Kembou Tsofack et al., 2017), histopathology (Eyngor et al., 2014; Tattiyapong et al., 2017),
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together with the relevant clinical signs and history of fish (Eyngor et al., 2014; Surachetpong et al., 2017). Although these techniques provide high sensitivity and specificity for TiLV
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detection, they have some limitations including requiring expensive equipment, well-trained personnel, and time to submit and process samples in a laboratory. Additionally, tilapia is cultured in developing countries where diagnostic resources and transportation access are
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limited. For these reasons, there is an urgent need to develop a rapid, accurate, and highly sensitive method that could be applied for on-farm diagnosis. loop-mediated
isothermal amplification
(LAMP)
technique
is
an isothermal
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amplification of nucleic acid at a constant temperature of 60-65°C (Notomi et al., 2000). Generally, a set of primers is commonly developed that can amplify six to eight different regions of the target pathogen genome. The assay is sensitive and rapid, yet so easy that the
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result can be interpreted within 30 to 60 min. Additionally, analysis of LAMP result relies on a change in the turbidity or colorimetric change in the reaction allowing untrained personnel to interpret the results. To detect an RNA virus, various reverse transcription LAMP (RTLAMP) assays have been developed to detect fish RNA viral pathogens. For example, a colorimetric RT-LAMP protocol was developed for the detection of nervous necrosis virus in olive flounder (Paralichthys olivaceus) with higher sensitivity than the nested RT-PCR and RT-PCR assays (Suebsing, et al., 2012). Similarly, a developed RT-LAMP protocol for infectious hematopoietic necrosis virus in Oncorhynchus keta demonstrated a low detection limit of 0.01 fg of viral RNA compared to 1 fg for nested RT-PCR (Zhang et al., 2014). Altogether, RT-LAMP assay has been validated as an efficient tool to detect disease
ACCEPTED MANUSCRIPT occurrence of aquatic animal pathogens; however, no LAMP assay has been developed specifically to detect TiLV disease in fish. The aim of the present study was to develop a rapid, sensitive, and accurate RT-LAMP assay for the detection of TiLV in fish tissues. The newly developed RT-LAMP protocol was applied to screen TiLV in infected cell culture materials, moribund and normal fish tissues collected from field samples. Moreover, the RT-LAMP protocol offers reliable sensitivity
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and specificity compared to RT-qPCR protocol for TiLV detection.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Experimental design and animal protocol In total, 166 tissue samples (liver and mucus) were collected from field outbreaks of high mortality associated with TiLV infection. The samples were collected from 14 farms in different regions of Thailand from 2015 to 2018. The moribund fish and healthy fish were
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collected from the same farms in which TiLV had been confirmed using conventional PCR or RT-qPCR after the collection process. The animal use protocol and sample collection had
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been extensively reviewed and approved by Kasetsart University Animal Use Committee
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(protocol number ACKU61-VET-009). 2.2 RT-LAMP primer design
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The segment 3 of TiLV originating from Thailand was retrieved from GenBank accession number KX631923 (Tilapia lake virus isolate TV1 segment 3). The set of primers
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composing internal primers (TiLV-FIP and TiLV-BIP) and external primers (TiLV-F3 and TiLV-B3) were designed using the Primer Explorer v.4 software (http://primerexplorer.jp/e/).
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The position and sequence of primers are shown in Fig 1 and Table 1. 2.3 RNA isolation and cDNA synthesis
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Briefly, 30 mg of liver tissues or mucus were collected in a microcentrifuge and ground in 1 mL Trizol reagent (Invitrogen, Carlsbad, CA, USA) using a hand-held homogenizer. For infected cell culture materials, 1 mL of L-15 medium containing E-11 cells (with or without
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TiLV inoculation) was transferred into a microcentrifuge tube. Then, the homogenized samples were processed for total RNA extraction according to the manufacturer’s protocol. The total RNA concentration was measured using a spectrophotometer (NanoDrop2000; Thermo Fisher Scientific Corp.; Carlsbad, CA, USA) and diluted with nuclease-free water to a final concentration of 100 ng/µL. The samples were immediately used or kept at -20 ºC for further analysis. 2.4 RT-LAMP and LAMP reaction The LAMP assay was performed in a 25 µL total volume of reaction mixture consisting of 1
SD II reaction buffer (Biotechrabbit; Berlin, Germany), 6 mM of MgSO4, 1.4 mM
dNTP set (Bioline; London, UK), 0.8 M of betaine (Sigma-Aldrich; St. Louis, MO, USA),
ACCEPTED MANUSCRIPT 0.052 mM of calcein mixture (Merck; Darmstadt Germany), 0.2 µM each of TiLV- FIP and TiLV-BIP primers, 1.6 µM each of TiLV-F3 and TILV-B3 primers, 0.32 U of Bst DNA polymerase and 5.8 µL of nuclease-free water. Finally, 3 µL of extracted RNA sample were added into the reaction tube. The sample was incubated at 65 ºC for 60 min and then at 80 ºC for 10 min. The RT-LAMP reaction mixture consisting of the same reagents and conditions as described in the LAMP protocol except for 0.1U of AMV reverse transcriptase (Promega;
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Madison, WI, USA) was added and nuclease-free water was used for adjustment. In addition, distilled water was used as the negative control in both techniques. The LAMP and RT-
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analyzed using an agarose gel electrophoresis technique.
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LAMP products were visualized by naked eyes for any colorimetric change and additionally
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2.5 Analysis of the RT-LAMP and LAMP products using gel detection and calcein detection A volume of 2.5 µL of RT-LAMP and LAMP product was separated on 2% agarose gel
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electrophoresis (100 V constant for 40 min) in 0.5x Tris-borate EDTA buffer stained with Neogreen, DNA staining reagent (NeoScience Co. Ltd.; Suwon, South Korea). On validation of the positive and negative controls (including the samples without TiLV as the negative
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samples), the samples were considered positive if they showed a characteristic ladder-like pattern. In addition, a solution that turned green was a positive sample based on visual
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inspection.
2.6 Reverse transcription quantitative polymerase chain reaction
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Each RT-qPCR assay was performed in a total of 10 µL of reaction containing 5 µL iTaqTM Universal SYBR Green supermix (2x), 0.3 µL of TiLV-112F forward primer and TiLV-112R reverse primer (each with a 10 pmol concentration), 0.4 µL of nuclease-free water and 4 µL of cDNA from positive samples and negative samples. The nucleotide sequences of primers TiLV-112F and TiLV-112R are indicated in Table 1. Subsequently, the reaction mixture was conducted in a qPCR thermocycler (CFX96T M Thermal Cycler; BioRad; Foster City, CA, USA) with conditions following the published protocol (Tattiyapong et al., 2018). 2.7 Sensitivity and specificity of RT-LAMP and RT-qPCR
ACCEPTED MANUSCRIPT The sensitivity of RT-LAMP and RT-qPCR was determined using a 10-fold serial dilution of RNA extracted from infected tissues or E-11 cells. The concentration of RNA ranged from 100 ng to 10 fg. The sensitivity of the RT-LAMP assays was evaluated using agarose gel electrophoresis while the RT-qPCR result was analyzed as previously described (Tattiyapong et al., 2018). The specificity of the RT-LAMP protocol was tested against RNA samples (100 ng) extracted from uninfected E-11 cells or infected fish tissues with other
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important pathogenic bacteria and viruses in tilapia, including Streptococcus agalactiae,
3. Results and Discussion
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3.1 Specificity of RT-LAMP for TiLV detection
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Flavobacterium columnare, Fransicella noatunensis, Aeromonas hydrophila, and Iridovirus.
In this study, a set of primers was designed from the target nucleotide sequence of segment 3
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of TiLV isolated from tilapia in Thailand (Surachetpong et al., 2017). The nucleotide sequences and positions of the four primer sets are shown in Fig. 1 and Table 1. The specificity of the LAMP primers was evaluated using total RNA samples prepared from the
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liver and mucus of TiLV-infected fish, and uninfected fish. As shown in Fig. 2A., a ladderlike pattern product was visualized in the liver and mucus of TiLV-infected samples on
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agarose gel while no band was observed in uninfected samples. The positive ladder-like pattern was the result of multiple amplifications of inverted DNA repeats which form stemloop DNA structures with various sizes of PCR products in the final reaction (Notomi et al.,
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2015; Notomi et al., 2000). Consistently, a colorimetric change of a fluorescent dye from light yellow to fluorescent green was observed in TiLV-infected samples, but no color change was detected in uninfected-TiLV samples (Fig. 2A and Supplementary Fig. 2A). The formation of the PCR product in the positive sample caused the calcein dye, a metal ionbinding fluorophore, to form an insoluble salt complex (manganese-pyrophosphate), making the reaction fluorescent and hence observable under UV light at 365 nm exposure wavelength. Thus, the interpretation of the RT-LAMP assay could be visualized by the naked eye without requiring expensive equipment or trained laboratory personnel. Furthermore, to assess the cross-reactivity of the LAMP primers, infected tissues were also tested with pathogenic
bacteria
and
viruses: Streptococcus agalactiae,
Francisella
noatulensis,
Flavobacterium columnare, Aeromonas hydrophila and Iridovirus. No ladder-like pattern or
ACCEPTED MANUSCRIPT color change was observed in these samples, indicating no cross-reactivity of the RT-LAMP assays (Fig. 2B). 3.2 Comparison of RT-LAMP and RT-qPCR assay Next, the newly developed RT-LAMP assay was compared to the recently established RT-qPCR protocol (Tattiyapong et al., 2018). A serial ten-fold dilution of the RNA from
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liver samples prepared from TiLV infected fish tissues was used as a template to assess the sensitivity of the RT-qPCR and RT-LAMP assays. The amplification curve and the intensity
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of amplified products on agarose gel revealed that the RT-qPCR assay could detect TiLV genomic RNA in 10 fg RNA sample (Fig. 3). Likewise, the colorimetric change of RT-
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LAMP reaction could be observed in tubes containing RNA samples ranging from 100 ng to 100 fg. Additionally, comparison of the sensitivity of viral isolation in cell culture, RT-
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LAMP, and RT-qPCR assays using infected cell culture materials revealed that the RTLAMP and RT-qPCR methods had comparable detection limit with 10 times more sensitive
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than the viral isolation in cell culture (Supplementary Table 1 and Supplementary Fig. 2.).
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3.3 Validation of RT-LAMP for TiLV detection
The reliability and applicability of RT-LAMP assay was tested using 5 infected and 5
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uninfected cell culture materials, and 166 tissue samples collected from TiLV-infected fish and uninfected fish from tilapia farms in Thailand with a history of TiLV outbreaks. Four groups of samples were examined from fish samples and cell culture materials: 1) liver RNA,
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2) mucus RNA, 3) liver cDNA, and 4) E-11 RNA. Among these samples, 88 were confirmed to be TiLV-positive based on the result of the RT-qPCR technique with the cut-off Ct value at 34 (Tattiyapong et al., 2018). Analysis of TiLV-infected RNA using RT-LAMP revealed positive results in 80% of the liver samples (36/45 samples), 35% of the mucus samples (7/20 samples), and 100% of the infected E-11 cells (5/5 samples) compared to the RT-qPCR assay (Table 2). For the 88 RNA and cDNA samples confirmed TiLV negative, there was no fluorescent change in any tested sample, indicating no false positive results using RT-LAMP for uninfected samples. Notably, the low sensitivity of RT-LAMP was observed in the mucus RNA samples compared to RNA prepared from infected liver. Although mucus could serve as a non-lethal sampling process for TiLV detection (Liamnimitr et al., 2018), due to the low sensitivity of
ACCEPTED MANUSCRIPT the RT-LAMP assay, it is suggested to use liver tissues for better accuracy. It is possible that the viral genomic RNA in mucus samples degraded more rapidly than the genomic RNA in liver tissues, as these samples were collected and preserved in the refrigerator for a certain period. It has been shown that preservation of fish tissues under freezing condition affects the quality and quantity of the virus (Thammatorn et al., 2019). In general, cDNA is more stable and easier to preserve than the vulnerable RNA. Therefore, the degradation of RNA could lead to misinterpreted diagnosis. In this study, the
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cDNA samples were included in the analysis for comparison with the RNA samples to test this LAMP assay. The results showed a positive detection of TiLV in cDNA liver samples at
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83.33% (15/18 samples), which was comparable to the level achieved using RNA liver
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samples (80%). This result suggested that both the cDNA and RNA liver samples had similar levels of detection using the protocol developed in this study. Besides tilapia, the LAMP
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assay can be applied for TiLV screening in intermediate hosts or carriers as recent study
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found that TiLV can cause disease in other fish species (Jaemwimol et al., 2018).
4. Conclusion
Practically, a desirable diagnostic test is an assay with good sensitivity, specificity,
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simplicity, and immediate result interpretation. This study developed an RT-LAMP protocol for the detection of TiLV in fish tissues and cell lines. Compared with RT-qPCR, the RT-
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LAMP assay offered high sensitivity and reliability in the diagnosis of TiLV infection. Moreover, the RT-LAMP assay could be applied to detect the disease in resource-limited
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countries where there is an urgent need for rapid diagnostic assay to guide TiLV control.
Acknowledgements
The project was supported 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.
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Figure Legends Fig 1. Target nucleotide sequence positions for RT-LAMP primers obtained from segment 3 of TiLV: (1) TiLV-F3, (2) TiLV-FIP (F1c), (3) TiLV-FIP (F2), (4) TiLV-BIP (B1c), (5) TiLV-BIP (B2), (6) TiLV-B3
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Fig 2. Specificity of RT-LAMP assay for TiLV detection in fish tissues visualized using gel electrophoresis and colorimetric change. (A) Amplification products of TiLV-infected tissues and TiLV-uninfected tissues; M = 1 Kb DNA ladder, 1-3 = Liver of TiLV-infected fish, 4-6 = Mucus of TiLV-infected fish, 7-9 = Liver of TiLV-uninfected fish, 10-12 = Mucus of TiLVuninfected fish. (B) Amplification products of fish tissues infected with other bacteria or
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viruses; M = 1 Kb DNA ladder, 1-5 = Tilapia tissues positive to bacteria and viruses: Streptococcus agalactiae, Francisella noatunensis, Flavobacterium columnare, Aeromonas
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hydrophila, and Iridovirus.
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Fig 3. Comparison of sensitivity of RT-LAMP and RT-qPCR protocols. RNA template prepared from liver of TiLV-infected fish was ten-fold serially diluted from 100 ng to 10 fg folds (lanes 1–8, in order). (A) Specific band product (112 bp) of the RT-qPCR reaction was
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separated in agarose gel, M = Ultra low range DNA marker. (B) Colorimetric visualization of
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RT-LAMP products
Supplementary Fig 1. Specificity of RT-LAMP assay for TiLV detection in cell culture materials. Amplification products and color changes of RT-LAMP products of infected E-11 cells (lane 1-3), and uninfected E-11 cells (lane 4-6), M = 1 Kb DNA ladder
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Supplementary Fig 2. Colorimetric visualization of RT-LAMP products from a ten-fold serially diluted (10-1 to 10-7 ; tube 1 to 7) of RNA template from infected E-11 cells. Tube 8 = no template control. Table 1 Nucleotide sequences of primers for RT-LAMP and RT-qPCR analysis of tilapia lake virus (TiLV) used in this study Purpose RT-LAMP Forward outer primer
Positiona 319-336
Sequence (53) GGGCACAAGGCATCCTAC
TiLV-B3
RT-LAMP Backward outer primer RT-LAMP Forward inner primer (F1c-F2)
547-530
AGACCACACTCCTCACCG
F1c: 413-432 F2: 370-384
CGATACAAGGCTTCGGGCCGGATG CTGAGCTGAGGGAAC
RT-LAMP B1c: 437-456 Backward inner B2: 497-511 primer (B1c-B2)
GGTGGCACCACCCAGACTTGAGAG CACTCGAAGAACCCA
TiLV-FIP
TiLV- BIP
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Primer name TiLV-F3
ACCEPTED MANUSCRIPT TiLV-112F
RT-qPCR Forward primer
478-502
CTGAGCTAAAGAGGCAATATGGATT
TiLV-112R
RT-qPCR 563-589 CGTGCGTACTCGTTCAGTATAAGTTCT Reverse primer a Nucleotide sequences correspond to the positions of segment 3 of TiLV (Genbank accession number KX631923)
Description
Type of nucleic acid
Number. of samples
Infected materials
Liver
RNA
45
Mucus
RNA
20
Liver
cDNA
18
E-11 cells
RNA
Liver
RNA
Mucus
RNA
20
Liver
cDNA
18
RNA
5
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LAMP (%) (positive/total sample) 80.00 (36/45) 35.00 (7/20) 83.33 (15/18) 100 (5/5)
0 (0/45) 0 (0/20) 0 (0/18) 0 (0/5)
0 (0/45) 0 (0/20) 0 (0/18) 0 (0/5)
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E-11 cells
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Uninfected materials
5
RT-qPCR (%) (positive/total sample) 100 (45/45) 100 (20/20) 100 (18/18) 100 (5/5)
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Type of sample
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Table 2 Detection of TiLV in fish and cell culture samples using LAMP and RT-qPCR
ACCEPTED MANUSCRIPT Highlights:
RT-LAMP method has been developed for the detection of tilapia lake virus in tilapia.
Four primer sets were designed to amplify TiLV in field collected tissue samples.
Comparison of RT-qPCR and RT-LAMP protocols revealed a comparable specific
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RT-LAMP provides rapid and sensitive tool for on farm detection of TiLV.
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and sensitive for TiLV detection.
Figure 1
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Figure 3
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Theerawut Phusantisampan, Puntanat Tattiyapong, Palita Mutrakulcharoen, Malinee Sriariyanun, Win Surachetpong PII: DOI: Reference:
S0044-8486(18)32808-4 https://doi.org/10.1016/j.aquaculture.2019.04.015 AQUA 634049
To appear in:
aquaculture
Received date: Revised date: Accepted date:
24 December 2018 21 February 2019 3 April 2019
Please cite this article as: T. Phusantisampan, P. Tattiyapong, P. Mutrakulcharoen, et al., Rapid detection of tilapia lake virus using a one-step reverse transcription loopmediated isothermal amplification assay, aquaculture, https://doi.org/10.1016/ j.aquaculture.2019.04.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Rapid detection of tilapia lake virus using a one-step reverse transcription loopmediated isothermal amplification assay Theerawut Phusantisampan1 , Puntanat Tattiyapong2,3 , Sriariyanun4 , Win Surachetpong2,3 *
Palita Mutrakulcharoen4 ,
Malinee
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Department of Biotechnology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok (KMUTNB), Thailand. 2
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Department of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Thailand. 3
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Center for Advanced Studies for Agriculture and Food, Kasetsart University, Institute for Advanced Studies, Kasetsart University, Bangkok 10900, Thailand (CASAF, NRU-KU), Thailand.
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The Sirindhorn Thai-German International Graduate School of Engineering (TGGS), King Mongkut's University of Technology North Bangkok (KMUTNB), Thailand.
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Abstract
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Tilapia lake virus (TiLV) is a newly emerging viral disease in tilapia with recent outbreaks in many parts of the world. There is an urgent need to develop an accurate, sensitive, on-farm method for rapid detection of TiLV to limit economic loss and prevent the
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spread of the virus into new geographical areas. In this study, we developed a rapid, one-step reverse transcription, loop-mediated, isothermal amplification (RT-LAMP) method for the
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detection of TiLV in fish tissues. Our results revealed that the RT-LAMP assay was able to detect TiLV infection in infected cell culture materials, and fish samples collected from different geographic locations in Thailand. In total, 166 tissue samples were collected from TiLV-infected fish and uninfected fish from tilapia farms in Thailand with a history of TiLV
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outbreaks, and fish showing clinical signs of TiLV infection were tested using our RT-LAMP protocol. The RT-LAMP assay offered high specificity and sensitivity compared to reverse transcription polymerase chain reaction (RT-qPCR) assay. These results supported the application of the RT-LAMP assay for the diagnosis of TiLV with benefits including reduced analysis time and easy interpretation of results based on the colorimetric change, thus offering a diagnostic tool in resource-limited countries where there is an urgent need for rapid diagnostic assay to guide TiLV control. Keywords: RT-LAMP, tilapia lake virus, diagnosis, tilapia Corresponding author: [email protected]
ACCEPTED MANUSCRIPT 1. Introduction Tilapia lake virus disease (TiLVD), caused by the tilapia lake virus (TiLV), is an emerging viral disease which has been recently reported in countries where tilapia is the main aquaculture species. The virus has been detected in wild and farm-raised tilapia and is associated with high mortality such as in summer mortality syndrome (Nicholson et al., 2017), one-month mortality syndrome (Surachetpong et al., 2017) and syncytial hepatitis
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disease (Del-Pozo et al., 2017). Currently, diagnosis of TiLV infection has relied on the detection of viral genomic RNA using reverse transcription polymerase chain reaction (RT-
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PCR) (Dong et al., 2017; Eyngor et al., 2014; Kembou Tsofack et al., 2017), RT-quantitative PCR (RT-qPCR) (Kembou Tsofack et al., 2017; Nicholson et al., 2018; Tattiyapong et al.,
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2018; Waiyamitra et al., 2018), virus isolation in the cell culture (Eyngor et al., 2014; Kembou Tsofack et al., 2017), histopathology (Eyngor et al., 2014; Tattiyapong et al., 2017),
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together with the relevant clinical signs and history of fish (Eyngor et al., 2014; Surachetpong et al., 2017). Although these techniques provide high sensitivity and specificity for TiLV
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detection, they have some limitations including requiring expensive equipment, well-trained personnel, and time to submit and process samples in a laboratory. Additionally, tilapia is cultured in developing countries where diagnostic resources and transportation access are
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limited. For these reasons, there is an urgent need to develop a rapid, accurate, and highly sensitive method that could be applied for on-farm diagnosis. loop-mediated
isothermal amplification
(LAMP)
technique
is
an isothermal
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amplification of nucleic acid at a constant temperature of 60-65°C (Notomi et al., 2000). Generally, a set of primers is commonly developed that can amplify six to eight different regions of the target pathogen genome. The assay is sensitive and rapid, yet so easy that the
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result can be interpreted within 30 to 60 min. Additionally, analysis of LAMP result relies on a change in the turbidity or colorimetric change in the reaction allowing untrained personnel to interpret the results. To detect an RNA virus, various reverse transcription LAMP (RTLAMP) assays have been developed to detect fish RNA viral pathogens. For example, a colorimetric RT-LAMP protocol was developed for the detection of nervous necrosis virus in olive flounder (Paralichthys olivaceus) with higher sensitivity than the nested RT-PCR and RT-PCR assays (Suebsing, et al., 2012). Similarly, a developed RT-LAMP protocol for infectious hematopoietic necrosis virus in Oncorhynchus keta demonstrated a low detection limit of 0.01 fg of viral RNA compared to 1 fg for nested RT-PCR (Zhang et al., 2014). Altogether, RT-LAMP assay has been validated as an efficient tool to detect disease
ACCEPTED MANUSCRIPT occurrence of aquatic animal pathogens; however, no LAMP assay has been developed specifically to detect TiLV disease in fish. The aim of the present study was to develop a rapid, sensitive, and accurate RT-LAMP assay for the detection of TiLV in fish tissues. The newly developed RT-LAMP protocol was applied to screen TiLV in infected cell culture materials, moribund and normal fish tissues collected from field samples. Moreover, the RT-LAMP protocol offers reliable sensitivity
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and specificity compared to RT-qPCR protocol for TiLV detection.
ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Experimental design and animal protocol In total, 166 tissue samples (liver and mucus) were collected from field outbreaks of high mortality associated with TiLV infection. The samples were collected from 14 farms in different regions of Thailand from 2015 to 2018. The moribund fish and healthy fish were
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collected from the same farms in which TiLV had been confirmed using conventional PCR or RT-qPCR after the collection process. The animal use protocol and sample collection had
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been extensively reviewed and approved by Kasetsart University Animal Use Committee
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(protocol number ACKU61-VET-009). 2.2 RT-LAMP primer design
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The segment 3 of TiLV originating from Thailand was retrieved from GenBank accession number KX631923 (Tilapia lake virus isolate TV1 segment 3). The set of primers
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composing internal primers (TiLV-FIP and TiLV-BIP) and external primers (TiLV-F3 and TiLV-B3) were designed using the Primer Explorer v.4 software (http://primerexplorer.jp/e/).
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The position and sequence of primers are shown in Fig 1 and Table 1. 2.3 RNA isolation and cDNA synthesis
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Briefly, 30 mg of liver tissues or mucus were collected in a microcentrifuge and ground in 1 mL Trizol reagent (Invitrogen, Carlsbad, CA, USA) using a hand-held homogenizer. For infected cell culture materials, 1 mL of L-15 medium containing E-11 cells (with or without
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TiLV inoculation) was transferred into a microcentrifuge tube. Then, the homogenized samples were processed for total RNA extraction according to the manufacturer’s protocol. The total RNA concentration was measured using a spectrophotometer (NanoDrop2000; Thermo Fisher Scientific Corp.; Carlsbad, CA, USA) and diluted with nuclease-free water to a final concentration of 100 ng/µL. The samples were immediately used or kept at -20 ºC for further analysis. 2.4 RT-LAMP and LAMP reaction The LAMP assay was performed in a 25 µL total volume of reaction mixture consisting of 1
SD II reaction buffer (Biotechrabbit; Berlin, Germany), 6 mM of MgSO4, 1.4 mM
dNTP set (Bioline; London, UK), 0.8 M of betaine (Sigma-Aldrich; St. Louis, MO, USA),
ACCEPTED MANUSCRIPT 0.052 mM of calcein mixture (Merck; Darmstadt Germany), 0.2 µM each of TiLV- FIP and TiLV-BIP primers, 1.6 µM each of TiLV-F3 and TILV-B3 primers, 0.32 U of Bst DNA polymerase and 5.8 µL of nuclease-free water. Finally, 3 µL of extracted RNA sample were added into the reaction tube. The sample was incubated at 65 ºC for 60 min and then at 80 ºC for 10 min. The RT-LAMP reaction mixture consisting of the same reagents and conditions as described in the LAMP protocol except for 0.1U of AMV reverse transcriptase (Promega;
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Madison, WI, USA) was added and nuclease-free water was used for adjustment. In addition, distilled water was used as the negative control in both techniques. The LAMP and RT-
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analyzed using an agarose gel electrophoresis technique.
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LAMP products were visualized by naked eyes for any colorimetric change and additionally
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2.5 Analysis of the RT-LAMP and LAMP products using gel detection and calcein detection A volume of 2.5 µL of RT-LAMP and LAMP product was separated on 2% agarose gel
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electrophoresis (100 V constant for 40 min) in 0.5x Tris-borate EDTA buffer stained with Neogreen, DNA staining reagent (NeoScience Co. Ltd.; Suwon, South Korea). On validation of the positive and negative controls (including the samples without TiLV as the negative
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samples), the samples were considered positive if they showed a characteristic ladder-like pattern. In addition, a solution that turned green was a positive sample based on visual
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inspection.
2.6 Reverse transcription quantitative polymerase chain reaction
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Each RT-qPCR assay was performed in a total of 10 µL of reaction containing 5 µL iTaqTM Universal SYBR Green supermix (2x), 0.3 µL of TiLV-112F forward primer and TiLV-112R reverse primer (each with a 10 pmol concentration), 0.4 µL of nuclease-free water and 4 µL of cDNA from positive samples and negative samples. The nucleotide sequences of primers TiLV-112F and TiLV-112R are indicated in Table 1. Subsequently, the reaction mixture was conducted in a qPCR thermocycler (CFX96T M Thermal Cycler; BioRad; Foster City, CA, USA) with conditions following the published protocol (Tattiyapong et al., 2018). 2.7 Sensitivity and specificity of RT-LAMP and RT-qPCR
ACCEPTED MANUSCRIPT The sensitivity of RT-LAMP and RT-qPCR was determined using a 10-fold serial dilution of RNA extracted from infected tissues or E-11 cells. The concentration of RNA ranged from 100 ng to 10 fg. The sensitivity of the RT-LAMP assays was evaluated using agarose gel electrophoresis while the RT-qPCR result was analyzed as previously described (Tattiyapong et al., 2018). The specificity of the RT-LAMP protocol was tested against RNA samples (100 ng) extracted from uninfected E-11 cells or infected fish tissues with other
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important pathogenic bacteria and viruses in tilapia, including Streptococcus agalactiae,
3. Results and Discussion
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3.1 Specificity of RT-LAMP for TiLV detection
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Flavobacterium columnare, Fransicella noatunensis, Aeromonas hydrophila, and Iridovirus.
In this study, a set of primers was designed from the target nucleotide sequence of segment 3
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of TiLV isolated from tilapia in Thailand (Surachetpong et al., 2017). The nucleotide sequences and positions of the four primer sets are shown in Fig. 1 and Table 1. The specificity of the LAMP primers was evaluated using total RNA samples prepared from the
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liver and mucus of TiLV-infected fish, and uninfected fish. As shown in Fig. 2A., a ladderlike pattern product was visualized in the liver and mucus of TiLV-infected samples on
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agarose gel while no band was observed in uninfected samples. The positive ladder-like pattern was the result of multiple amplifications of inverted DNA repeats which form stemloop DNA structures with various sizes of PCR products in the final reaction (Notomi et al.,
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2015; Notomi et al., 2000). Consistently, a colorimetric change of a fluorescent dye from light yellow to fluorescent green was observed in TiLV-infected samples, but no color change was detected in uninfected-TiLV samples (Fig. 2A and Supplementary Fig. 2A). The formation of the PCR product in the positive sample caused the calcein dye, a metal ionbinding fluorophore, to form an insoluble salt complex (manganese-pyrophosphate), making the reaction fluorescent and hence observable under UV light at 365 nm exposure wavelength. Thus, the interpretation of the RT-LAMP assay could be visualized by the naked eye without requiring expensive equipment or trained laboratory personnel. Furthermore, to assess the cross-reactivity of the LAMP primers, infected tissues were also tested with pathogenic
bacteria
and
viruses: Streptococcus agalactiae,
Francisella
noatulensis,
Flavobacterium columnare, Aeromonas hydrophila and Iridovirus. No ladder-like pattern or
ACCEPTED MANUSCRIPT color change was observed in these samples, indicating no cross-reactivity of the RT-LAMP assays (Fig. 2B). 3.2 Comparison of RT-LAMP and RT-qPCR assay Next, the newly developed RT-LAMP assay was compared to the recently established RT-qPCR protocol (Tattiyapong et al., 2018). A serial ten-fold dilution of the RNA from
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liver samples prepared from TiLV infected fish tissues was used as a template to assess the sensitivity of the RT-qPCR and RT-LAMP assays. The amplification curve and the intensity
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of amplified products on agarose gel revealed that the RT-qPCR assay could detect TiLV genomic RNA in 10 fg RNA sample (Fig. 3). Likewise, the colorimetric change of RT-
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LAMP reaction could be observed in tubes containing RNA samples ranging from 100 ng to 100 fg. Additionally, comparison of the sensitivity of viral isolation in cell culture, RT-
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LAMP, and RT-qPCR assays using infected cell culture materials revealed that the RTLAMP and RT-qPCR methods had comparable detection limit with 10 times more sensitive
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than the viral isolation in cell culture (Supplementary Table 1 and Supplementary Fig. 2.).
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3.3 Validation of RT-LAMP for TiLV detection
The reliability and applicability of RT-LAMP assay was tested using 5 infected and 5
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uninfected cell culture materials, and 166 tissue samples collected from TiLV-infected fish and uninfected fish from tilapia farms in Thailand with a history of TiLV outbreaks. Four groups of samples were examined from fish samples and cell culture materials: 1) liver RNA,
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2) mucus RNA, 3) liver cDNA, and 4) E-11 RNA. Among these samples, 88 were confirmed to be TiLV-positive based on the result of the RT-qPCR technique with the cut-off Ct value at 34 (Tattiyapong et al., 2018). Analysis of TiLV-infected RNA using RT-LAMP revealed positive results in 80% of the liver samples (36/45 samples), 35% of the mucus samples (7/20 samples), and 100% of the infected E-11 cells (5/5 samples) compared to the RT-qPCR assay (Table 2). For the 88 RNA and cDNA samples confirmed TiLV negative, there was no fluorescent change in any tested sample, indicating no false positive results using RT-LAMP for uninfected samples. Notably, the low sensitivity of RT-LAMP was observed in the mucus RNA samples compared to RNA prepared from infected liver. Although mucus could serve as a non-lethal sampling process for TiLV detection (Liamnimitr et al., 2018), due to the low sensitivity of
ACCEPTED MANUSCRIPT the RT-LAMP assay, it is suggested to use liver tissues for better accuracy. It is possible that the viral genomic RNA in mucus samples degraded more rapidly than the genomic RNA in liver tissues, as these samples were collected and preserved in the refrigerator for a certain period. It has been shown that preservation of fish tissues under freezing condition affects the quality and quantity of the virus (Thammatorn et al., 2019). In general, cDNA is more stable and easier to preserve than the vulnerable RNA. Therefore, the degradation of RNA could lead to misinterpreted diagnosis. In this study, the
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cDNA samples were included in the analysis for comparison with the RNA samples to test this LAMP assay. The results showed a positive detection of TiLV in cDNA liver samples at
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83.33% (15/18 samples), which was comparable to the level achieved using RNA liver
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samples (80%). This result suggested that both the cDNA and RNA liver samples had similar levels of detection using the protocol developed in this study. Besides tilapia, the LAMP
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assay can be applied for TiLV screening in intermediate hosts or carriers as recent study
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found that TiLV can cause disease in other fish species (Jaemwimol et al., 2018).
4. Conclusion
Practically, a desirable diagnostic test is an assay with good sensitivity, specificity,
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simplicity, and immediate result interpretation. This study developed an RT-LAMP protocol for the detection of TiLV in fish tissues and cell lines. Compared with RT-qPCR, the RT-
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LAMP assay offered high sensitivity and reliability in the diagnosis of TiLV infection. Moreover, the RT-LAMP assay could be applied to detect the disease in resource-limited
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countries where there is an urgent need for rapid diagnostic assay to guide TiLV control.
Acknowledgements
The project was supported 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.
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Figure Legends Fig 1. Target nucleotide sequence positions for RT-LAMP primers obtained from segment 3 of TiLV: (1) TiLV-F3, (2) TiLV-FIP (F1c), (3) TiLV-FIP (F2), (4) TiLV-BIP (B1c), (5) TiLV-BIP (B2), (6) TiLV-B3
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Fig 2. Specificity of RT-LAMP assay for TiLV detection in fish tissues visualized using gel electrophoresis and colorimetric change. (A) Amplification products of TiLV-infected tissues and TiLV-uninfected tissues; M = 1 Kb DNA ladder, 1-3 = Liver of TiLV-infected fish, 4-6 = Mucus of TiLV-infected fish, 7-9 = Liver of TiLV-uninfected fish, 10-12 = Mucus of TiLVuninfected fish. (B) Amplification products of fish tissues infected with other bacteria or
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viruses; M = 1 Kb DNA ladder, 1-5 = Tilapia tissues positive to bacteria and viruses: Streptococcus agalactiae, Francisella noatunensis, Flavobacterium columnare, Aeromonas
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hydrophila, and Iridovirus.
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Fig 3. Comparison of sensitivity of RT-LAMP and RT-qPCR protocols. RNA template prepared from liver of TiLV-infected fish was ten-fold serially diluted from 100 ng to 10 fg folds (lanes 1–8, in order). (A) Specific band product (112 bp) of the RT-qPCR reaction was
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separated in agarose gel, M = Ultra low range DNA marker. (B) Colorimetric visualization of
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RT-LAMP products
Supplementary Fig 1. Specificity of RT-LAMP assay for TiLV detection in cell culture materials. Amplification products and color changes of RT-LAMP products of infected E-11 cells (lane 1-3), and uninfected E-11 cells (lane 4-6), M = 1 Kb DNA ladder
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Supplementary Fig 2. Colorimetric visualization of RT-LAMP products from a ten-fold serially diluted (10-1 to 10-7 ; tube 1 to 7) of RNA template from infected E-11 cells. Tube 8 = no template control. Table 1 Nucleotide sequences of primers for RT-LAMP and RT-qPCR analysis of tilapia lake virus (TiLV) used in this study Purpose RT-LAMP Forward outer primer
Positiona 319-336
Sequence (53) GGGCACAAGGCATCCTAC
TiLV-B3
RT-LAMP Backward outer primer RT-LAMP Forward inner primer (F1c-F2)
547-530
AGACCACACTCCTCACCG
F1c: 413-432 F2: 370-384
CGATACAAGGCTTCGGGCCGGATG CTGAGCTGAGGGAAC
RT-LAMP B1c: 437-456 Backward inner B2: 497-511 primer (B1c-B2)
GGTGGCACCACCCAGACTTGAGAG CACTCGAAGAACCCA
TiLV-FIP
TiLV- BIP
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Primer name TiLV-F3
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RT-qPCR Forward primer
478-502
CTGAGCTAAAGAGGCAATATGGATT
TiLV-112R
RT-qPCR 563-589 CGTGCGTACTCGTTCAGTATAAGTTCT Reverse primer a Nucleotide sequences correspond to the positions of segment 3 of TiLV (Genbank accession number KX631923)
Description
Type of nucleic acid
Number. of samples
Infected materials
Liver
RNA
45
Mucus
RNA
20
Liver
cDNA
18
E-11 cells
RNA
Liver
RNA
Mucus
RNA
20
Liver
cDNA
18
RNA
5
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LAMP (%) (positive/total sample) 80.00 (36/45) 35.00 (7/20) 83.33 (15/18) 100 (5/5)
0 (0/45) 0 (0/20) 0 (0/18) 0 (0/5)
0 (0/45) 0 (0/20) 0 (0/18) 0 (0/5)
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E-11 cells
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Uninfected materials
5
RT-qPCR (%) (positive/total sample) 100 (45/45) 100 (20/20) 100 (18/18) 100 (5/5)
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Type of sample
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Table 2 Detection of TiLV in fish and cell culture samples using LAMP and RT-qPCR
ACCEPTED MANUSCRIPT Highlights:
RT-LAMP method has been developed for the detection of tilapia lake virus in tilapia.
Four primer sets were designed to amplify TiLV in field collected tissue samples.
Comparison of RT-qPCR and RT-LAMP protocols revealed a comparable specific
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RT-LAMP provides rapid and sensitive tool for on farm detection of TiLV.
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and sensitive for TiLV detection.
Figure 1
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Figure 5