ARTICLE
Utilization of loop-mediated isothermal amplification (LAMP) technology for detecting White Spot Syndrome Virus (WSSV) and Vibrio spp. in Litopenaeus vannamei in selected sites in the Philippines Amalea Dulcene D. Nicolasora1, Benedict A. Maralit4, Christopher Marlowe A. Caipang3, Mudjekeewis D. Santos4, Adelaida Calpe5, and Mary Beth B. Maningas*1,2 1
Research Center for the Natural and Applied Sciences, Thomas Aquinas Research Complex Biological Sciences Department, College of Science, University of Santo Tomas, Philippines 1008 3 School of Applied Science, Temasek Polytechnic, Singapore 4 Genetic Fingerprinting Laboratory, National Fisheries Research and Development Institute, 101 Mother Ignacia St. Quezon City 1103 Philippines 5 Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development - Department of Science and Technology, Los Banos, Laguna 2
S
hrimp disease outbreaks in the Philippines remain to be uncontrollable. This is compounded by the inaccessibility of disease diagnostics to most shrimp farmers. The loop-mediated isothermal amplification (LAMP) is a new technology that is used as a practical alternative for rapid detection of viral and bacterial pathogens. The method proves to be rapid, highly sensitive, and costeffective compared to other detection assays. In this study, LAMP protocols for the detection of the two most common shrimp pathogens, white spot syndrome virus (WSSV) and Vibrio spp., in the Philippines were developed. A temperature range of 55⁰C to 68⁰C for WSSV detection and 59⁰C to 67⁰C for Vibrio spp., and incubation periods of 45 minutes to 1 hour, were proven to be the suitable conditions for the LAMP assay. Using *Corresponding author Email Address:
[email protected] Submitted: August 12, 2013 Revised: June 30, 2014 Accepted: July 4, 2014 Published: September 10, 2014 Editor-in-charge: Eduardo A. Padlan Vol. 7 | No. 2 | 2014
these conditions, asymptomatic Litopenaeus vannamei samples from selected sites (Iloilo, Batangas, Bulacan, Laoag, and Leyte) were tested for WSSV. Samples which indicated WSSV infection were from Iloilo (89.47%), Batangas (30.00%), Bulacan (43.33%), and Leyte (75.00%), while shrimps from Laoag City (0.00%) tested negative. Likewise, the occurrence of Vibrio spp. was determined in shrimps sampled in Pangasinan and six bacterial DNA isolates of Vibrio spp. were identified. Moreover, conventional PCR and microbiological methods were performed along with the LAMP reaction for comparison and further confirmation. The results showed that the LAMP assay was faster and 10 times more sensitive than polymerase chain reaction in detecting WSSV and was more efficient than the traditional microbiological method in diagnosing vibriosis. Overall, the results indicated that a LAMP protocol, which is more convenient, highly sensitive, faster, and more practical, has been effectively utilized to detect WSSV and vibriosis in selected Philippine shrimp farms. KEYWORDS loop-mediated isothermal amplification, polymerase chain reaction, white spot syndrome virus, Vibrio spp., shrimps
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INTRODUCTION Shrimp is one of the most popular seafood in the world. Annual global production of shrimps reaches up to 4 million metric tons, one-third of which comes from the shrimp culture industry (FAO 2010). In most aquaculture-producing countries in Asia, shrimp is considered an economic asset (Caipang and Aguana 2011). The prevalence of shrimp pathogens, however, has contributed to a significant loss in production. In the Philippines, farmed shrimps have great potential for global economic profit. The country was a top shrimp exporter in the early 1990s, earning approximately US$ 300,000,000 during its peak. Similar to the global conditions, disease problems in the late 1990s caused a considerable decline in the country’s shrimp output (FAO 2006) and until now the industry is still trying ways to recover its great loss. Diseases due to luminous Vibrio spp. and the white spot syndrome virus (WSSV) are regarded as the two major causes of decline in shrimp production. However, established protocols for diagnosis and preventive measures for infection that are adapted to the Philippine setting are still lacking. Early disease diagnosis must be carried out so that immediate mitigating measures could be undertaken to prevent massive losses due to heavy mortality. The development of new methods for early pathogen detection can be an effective means of prevention. Since small-scale farms constitute a big bulk of the Philippine shrimp industry, a lowcost, rapid, and simple on-site disease diagnostic protocol needs to be established. To date, various molecular techniques have been developed to detect viral and bacterial pathogens. Conventional polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) are the most popular and widely used methods. However, several disadvantages, such as the need for an expensive thermal cycler, insufficient specificity, and low amplification are observed for these techniques (Mori et al. 2001). Cost effectiveness and practicality are the major concerns in creating protocols that can improve the quality of shrimp products. In the Philippines, methods for prevention and early detection of pathogens in shrimp aquaculture have already been designed. PCR (Tapay et al. 1999, Maralit et al. 2011, Caipang and Aguana 2011, Alenton and Maningas 2011), multiplex PCR (Castroverde et al. 2006), pathogenicity test, histopathology, and traditional microbiological techniques (Lavilla-Pitogo et al. 1998, de la Peña et al. 2007) are the commonly used methods for disease diagnosis in shrimp in the country. However, expensive and sophisticated equipment are required for the implementation of such techniques. They are laborious and difficult to apply during on-site testing. In addition, these procedures are quite inaccessible to a large number of small-scale shrimp farmers because they do not possess the financial capabilities that are required for such assays. Notomi et al. (2000) developed a technology called loopmediated isothermal amplification (LAMP) that can detect a wide array of pathogens. This assay allows amplification of nu310
cleic acid with high specificity under isothermal conditions. It can amplify target sequences up to 10 9 copies at 60°C to 65°C incubation in an hour or less. It relies on the strand displacement activity of Bst polymerase and a set of four specially designed primers that recognize six independent target sequences (Notomi et al. 2000). LAMP reaction proceeds at isothermal conditions that can be performed using only a water bath or heater block to maintain the required temperature, essentially eliminating the need for an expensive thermocycler, making it a practical and low-cost diagnostic tool. Given that shrimp aquaculture is faced with the constant threat of bacterial and viral pathogens, an early detection technique with the possibility of on-site application is necessary. LAMP is a potential molecular-diseases diagnostic tool for shrimp farms in the Philippines, since it is both effective and practical. Thus, this study aims to develop a LAMP protocol that will be beneficial, low-cost, and accessible for on-site application in most shrimp farms. This work makes use of the set of primers designed by Maralit et al. (2012) and Xu et al. (2012) which were tested at a wider temperature range. The LAMP primers were utilized for WSSV and Vibrio spp. detection on selected sites in the Philippines. METHODOLOGY Shrimp Collection Litopenaeus vannamei were purchased from wet markets and collected from shrimp farms at randomly selected sites in the Philippines. The shrimp farms and the origin of the shrimps bought from wet markets are identified in Table 1. For the following sites (Iloilo, Batangas, Bulacan, and Leyte), 15 shrimps were collected for WSSV detection and for Bolinao, Pangasinan, 15 shrimps were tested for Vibrio infection. For each site, 2 shrimps were separated for species identification. Samples were stored in an ice bucket and were transported to the Thomas Aquinas Research Complex of the University of Santo Tomas. Sampled shrimps were stored in a -80⁰C freezer until the commencement of the experiment. Table 1. Sampling Sites of the Study Site Batangas Nasugbu Calatagan
Date
Sample
October November
2011 2012
Farm sample Market sample
Hagonoy, Bulacan
October
2012
Farm sample and Market sample
San Jose, Leyte
February
2012
Market sample
Iloilo
August
2012
Farm sample Farm sample Farm sample
Laoag City, Ilocos Sur
December
2012
Market sample
Bolinao, Pangasinan
February
2013
Market Sample
Barkatok Dagat Roxas
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Table 2. LAMP Primers Primer Name WSSV C-FIP C-BIP C-F3 C-B3 WSSV-FIP WSSV-BIP WSSV-F3 WSSV-B3 Vibrio Vibrio-FIP Vibrio-BIP Vibrio-F3 Vibrio-B3
Sequence (5’– 3’) ACC CAA TGT ATG TGA CCA GCC -TTTT- GGA GGA GGT ACA TCC ACT ACA CTG GGT ACA GAT CAG GGA A -TTTT- ATT CAG ACC GCC CGT TAA GAG GAG GGT ACG GCA ATA CAA GGA TTC AAA ATT TAC TGT GG
Maralit et al. (2012)
GGG TCG TCG AAT GTT GCC CA -TTTT- GCC TAC GCA CCA ATC TGT G AAA GGA CAA TCC CTC TCC TGC G -TTTT- AGA ACG GAA GAA ACT GCC TT ACG GAG GAC CCA AAT CGA ACG GAG GAC CCA AAT CGA GCC TCT GCA ACA TCC TTT CC
Kono et al. (2004)
CGG CTG CTG GCA CGG AGT - TTTT-GCA TTA TTT GAC GTT AGC GAC AGA AG GAG CGT TAA TCG GAA TTA CTG GGC -TTTT- CCG GGC TTT CAC ATC TGA CTT AAC CAG TCG TGA GGA AGG TGG TGT CTA GTC TGC CAG TTT CAA ATG CT
DNA Extraction for WSSV Detection Tissue samples from gills, muscles, and intestines of 15 shrimps were dissected and stored separately in 1.5 mL sterile microcentrifuge tubes with appropriate labels. DNA was extracted from tissue samples using a Wizard Genomic DNA Purification Kit (Promega) following the manufacturer’s protocol. The DNA extracts were used for LAMP and PCR detection. Bacterial DNA Isolation for Vibrio spp. Fifteen shrimp samples were subjected to bacterial colony isolation. Dissected intestines of each shrimp were suspended in a 500 µL 0.9% natural sodium saline solution followed by homogenization. The lysate was streaked on a Thiosulfate Citrate Bile Salt (TCBS) agar plate and incubated for 24 to 48 hours at 25⁰C until bacterial growth was observed. Suspected Vibrio spp. colonies were subcultured and purified using MacConkey agar plates and incubated for 24 to 48 hour at 25⁰C. Pure isolates were cultured in 8 ml Tryptic Soy Broth media for 18 – 24 hours at 25⁰C and DNA extraction was conducted using a Wizard Genomic DNA Purification Kit (Promega) following the manufacturer’s protocol. Isolated DNA was subjected to PCR and LAMP detection using published Vibrio spp. primers and 16s rRNA bacterial primers. Molecular Analysis LAMP Detection The methodology that was used for LAMP detection was adapted from Maralit et al. (2012) with minor modifications. LAMP assay was carried out on a total of 25 µL reaction volume containing 2.0 pmol of each published FIP and –BIP inner primers (see Table 2), 0.2 pmol of -F3 and -B3 outer primers (see Table 2), 12.5 µL of 2X LAMP reaction buffer (40mM Tris–HCl, 20mM KCl, 16mM MgSO4, 20mM (NH4)2SO4, 0.1% Triton-X, 1.6M Betaine and 0.25 mM dNTPs each), 1 µL of target DNA, and 2.3 µL of distilled water. The mixture was initially incubated at 95° C for 5 minutes and then chilled on ice for 2 minutes. Subsequently, 0.3 U/ml of Bst DNA polymerase was added. The mixture was then incubated for 60 minutes at 55°C to 68°C for WSSV and 59°C to 67°C for Vibrio spp.; the reaction was terminated at 80°C for 10 minutes. Positive controls used were WSSV DNA template for viral detection and Vibrio vulnificus DNA isolate for bacterial detection. Reaction Vol. 7 | No. 2 | 2014
Sources
Xu et al. (2012)
mixtures without DNA template and non-Vibrio isolates (for Vibrio spp. detection) were used as negative controls. LAMP products were centrifuged briefly in a tabletop spinner and were run on a 2% agarose gel electrophoresis for confirmation. Further visualization was done by the addition of 4 µL of Sybr Safe stain (diluted 1000 times) to the LAMP products and illumination under UV light for color intensity observation. PCR Detection Detection with PCR was carried out with a 10 µL reaction mixture containing 1 unit of Taq polymerase, 0.6 µM of each published forward and reverse primers, 0.2 µM dNTP solution mix, 1 µL 10x buffer with 20 mM MgCl2, and 1 µL of DNA extract. Positive controls used were WSSV DNA template and Vibrio vulnificus DNA isolate, while a reaction mixture without any DNA template was used as a negative control. Thermal cycling conditions were: initial denaturation temperature of 95°C for 5 minutes, followed by 30 cycles of denaturation temperature of 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72° C for 30 seconds. Final DNA extension was at 72°C for 10 minutes, then 4°C was set as the holding temperature for storage. PCR products were viewed using agarose gel electrophoresis at 1% gel concentration. The primers used for PCR detection of WSSV and Vibrio spp. are shown in Table 3. Table 3. PCR Primers Primer Name
Sequence (5’– 3’)
WSSV WVF WVR
GTA CGG CAA TAC TGG AGG AGG GGA GAT GTG TAA GAT GGA CAA GG
Vibrio Vibrio-F3 Vibrio-B3
CAG TCG TGA GGA AGG TGG TGT CTA GTC TGC CAG TTT CAA ATG CT
16s 16s-F (27F) 16s-R (1492R)
AGA GTT TGA TCM TGG CTC AG ACC TTG TTA CGA CTT
Amplicon size/ Source 200 bp Maralit et al. (2011) 200 bp Xu et al. (2012) 1500 bp Lane et al. (1991) Universal primers
Agarose Gel Electrophoresis Agarose gel electrophoresis was carried out for the LAMP reaction by using 2% agarose gel with 3µL ethidium bromide (per 100 ml) for staining. Five microliters of
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LAMP products with 2 mL of 6X loading dye were loaded in each well. For the PCR detection, 1% agarose gel stained with 3 µL ethidium bromide loaded with 3 µL PCR products and 1 µL 6x loading dye was used. 1X TAE served as the running buffer and 25 minutes was the running time for LAMP products, while 20 minutes running time was used for PCR products. Data Interpretation and Calculations The percentage of occurrence of WSSV and Vibrio spp. in the shrimps collected from the selected sites was evaluated as the number of shrimps found positive for WSSV or Vibrio spp., divided by the total number of shrimps collected, multiplied by 100. RESULTS AND DISCUSSION LAMP Primer Selection and Optimization To establish a LAMP protocol for the WSSV and Vibrio spp. detections, primers that produced positive LAMP reactions using WSSV and Vibrio spp. isolates from the Philippines were selected and optimized at different temperature conditions.
Figure 1. LAMP detection at 65°C for 1 hour incubation period. WSSV positive templates were utilized. Primer set C (Maralit et al. 2012) and WSSV primer (Kono et. al. 2004) were being tested for primer selection for WSSV detection.
For WSSV detection, LAMP primers by Kono et al. (2004) and Maralit et al. (2012) were utilized (see Fig. 1). The primer set C of Maralit et al. (2012) yielded better results at 65⁰C incubation for 60 minutes. However, the published primers of Kono et al. (2004) gave false positive results in the LAMP assay due to the appearance of ladder-like patterns in the negative control. This finding indicates the tendency of the primers to interact with one another without the presence of a DNA template. Thus, the primer set C designed by Maralit et al. (2012) was chosen to be utilized for LAMP detection of WSSV on L. vannamei samples from the selected sites in the Philippines.
The LAMP reaction was carried out at 55⁰C, 63⁰C, and 68⁰C for 60 minutes using primer set C and WSSV DNA templates in order to determine the optimal temperature (Fig. 2). LAMP products were formed under those three conditions. The best result was obtained at 65⁰C incubation for 60 minutes (see Fig. 1). Reference sequences on which the primer design was based were also considered in the selection of primers. The WSSV sequence generated from the work of van Hulten et al. (2001) was the only reference sequence of Kono et al. (2004). However, primer set C was designed by Maralit et al. (2012) using PCR primers based on the conserved sequence of WSSV isolates from China, Taiwan, and Thailand, which also showed homology to WSSV DNA fragments sequenced from the Philippine isolate. This study validates the study made by Maralit et al. (2012) and shows that primer set C is the most suitable set of primers for WSSV LAMP detection in the Philippine setting. Published LAMP and PCR primers for identification of Vibrio spp. were mostly specific only to a certain strain. A universal primer for the genus has not been established, although the recent work of Xu et al. (2012) suggested a universal LAMP primer for pathogenic Vibrio spp. The LAMP primer set used in that study was designed from conserved regions of 16s rRNA specific to genus Vibrio and was determined to be specific to pathogenic Vibrio spp. The LAMP primer set was tested on 30 strains of nine Vibrio species and 21 related non-Vibrio microorganism strains as controls. A positive LAMP reaction was observed in the 30 pathogenic Vibrio strains, while no crossreaction was found on the control organisms, suggesting the specificity of the primers only to pathogenic Vibrio spp. (Xu et al. 2012). That led us to select and test those primers for the study of the suspected Vibrio bacterial DNA isolates. Using Vibrio vulnificus DNA as positive control, the optimum temperature of the selected primer was determined using the range of 55⁰C to 75⁰C conditions. As shown in Figure 3, the laddering pattern indicated a positive LAMP reaction. Products were generated at 59⁰C, 63⁰C, and 67⁰C and no product was formed at 55⁰C, 57⁰C, 71⁰C, 73.5⁰C, and 75⁰C (see Fig. 3). Thus LAMP detection was determined to be viable only in the 59⁰C to 67⁰C temperature set-ups.
Figure 3. Optimization of Vibrio spp. to varying temperature conditions. M - marker, A - 75⁰C, B - 73.5⁰C, C - 71⁰C, D - 67.2⁰C, E - 62.5⁰C, F - 58.9⁰C, G - 56.5⁰C, H - 55⁰C, (-) - negative control.
Figure 2. Optimization of primer set C at varying temperature conditions (55⁰C, 63⁰C, and 68⁰C) using WSSV DNA templates. 312
Specificity was evaluated using different bacterial DNA isolates and the findings in Figure 4 confirmed that the published Vibrio spp. primer was specific only to Vibrio species (Fig. 4,F). No bands or ladder-like patterns were produced on non-Vibrio bacterial DNA isolates.
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WSSV Detection To determine the sensitivity of LAMP compared to the conventional PCR method, WSSV detection using both methods was performed. Positive WSSV DNA template was serially diluted ten-fold. Results showed that LAMP, which detected the virus at 10-2 dilution with the concentration of 8.2 ng/µl, is ten (10) times more sensitive than conventional PCR, which was able to produce a band only down to 10 -1 dilution with a concentration of 84.6 ng/ml (see Fig. 5). Sybr Safe staining indicated that positive samples have greater intensity and were seen brighter than the negative control (see Fig. 6).
Figure 4. Primer specificity. LAMP primer set by Xu et al. (2012) specific to pathogenic Vibrio spp. was tested for specificity using non-Vibrio bacterial DNA isolates and Vibirio vulnificus DNA isolates. M - marker; A, B, C, D, E, and G non-Vibrio Bacterial DNA; F - Vibrio vulnificus DNA, H - negative control.
Temperature conditions wherein the selected primers were found to yield positive LAMP reactions were determined. Since the LAMP assay works under isothermal conditions, varying temperatures for incubation were utilized. The tested temperature range (55⁰C - 68⁰C) for the WSSV LAMP assay was found to be lower by five degrees and higher by 3 degrees than the published protocols of Notomi et al. (2000), Kono et al. (2004), and Maralit et al. (2012), which were limited only to 60⁰C 65⁰C incubation for an hour or less. Our results indicate that the formation of LAMP products is possible at a temperature lower than 60⁰C. However, the best product to be amplified was formed at 65⁰C and 60 minutes incubation, which are the suggested and established optimum parameters for WSSV LAMP detection (Maralit et al. 2012). According to Notomi et al. (2000), specificity increases at higher temperatures and that well -formed bands likewise indicate optimum conditions. Our optimization of Vibrio spp. primers produced results comparable to those observed in the study of Xu et al. (2012), where LAMP products were formed at 58⁰C to 66⁰C; and 62⁰C for 60 minutes was the optimum condition. An almost similar viable temperature range of 59⁰C to 67⁰C, and an optimum condition of 63⁰C for 60 minutes, were obtained in our study. Figure 5. Comparison of LAMP and PCR detection using ten-fold serial dilution of positive WSSV DNA templates.
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Figure 6. Sybr Safe staining of LAMP products for WSSV detection. Confirmation of WSSV positive samples by the addition of Sybr Safe stain on LAMP products and visualized under UV illuminator. 1, 3, and 5 – WSSV positive samples, 4 and 2 – negative control.
This result coincides with the findings of Yano et al. (2007) and Wang et al. (2008) for detection of E. coli and Salmonella. Also, Kono et al. (2004) showed that the LAMP assay detected WSSV DNA templates as low as 1 fg concentration, which is ten times more sensitive than nested PCR whose detection limit is 10 fg. Thus, LAMP has been proven to have high amplification efficiency, making it highly sensitive. Since, the reaction works under isothermal conditions, time loss during thermal change does not occur (Ren et al. 2008). Moreover, the high specificity of LAMP can also be attributed to its use of a set of four primers which targets six distinct regions in the DNA template, while PCR only targets a single region in the gene of interest (Notomi et al. 2000). WSSV LAMP detection was conducted on shrimps from the selected regions of the Philippines. Asymptomatic L. vannamei samples collected from the wet markets of various sampling sites indicated positivity to WSSV infection. Shrimps from Iloilo (89.47%), Batangas (30.00%), Bulacan (43.33%), and Leyte (75.00%) were found to be WSSV-infected, while shrimps from Laoag City tested negative for WSSV. A recent record of occurrence of WSSV is from the study of Maralit et al. (2011) where
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Figure 9. PCR detection results of Vibrio spp. using the five suspected Vibrio bacterial DNA isolates. 200 bp amplicons were produced for isolates identified as genus Vibrio spp. M – marker and V1 – V5 suspected Vibrio bacterial DNA isolates.
PCR was also conducted on suspected Vibrio spp. isolates using specific Vibrio primers, and a 200 bp amplicon was amplified from the samples (see Fig. 9). Figure 7. Prevalence of WSSV among selected sites in the Philippines. Litopenaeus vannamei from five randomly selected regions (Leyte, Iloilo, Batangas, Bulacan, and Laoag City) in the Philippines were tested for the presence or absence of WSSV using LAMP assay.
PCR was the method used for detection. In that study, the same species of shrimp (L. vannamei) was used and two sites were confirmed to be WSSV-positive, namely Zambales and General Santos City, while two other sites, Batangas and Capiz, were found to be negative. In our study, results verified one region as WSSV-free (Laoag City), three new regions as WSSV-infected (Iloilo, Bulacan, and Leyte); those three and the Batangas region, which was previously shown to be WSSV-free, are now added to the list of infected areas. Vibrio spp. Detection To assess the feasibility of LAMP as a practical tool for Vibrio spp. detection, it was compared to the conventional methods for bacterial detection. The TCBS (Fig. 8,B) agar plates utilized in the experiment, likewise, indicated growth of suspected Vibrio spp. colonies, which were observed as yellow-, green-, or blue- to green-centered colonies. The Triple Sugar Iron Agar (TSIA) biochemical test and API NE kit (bioMerieux, Inc.) identification were conducted for bacterial characterization, wherein 24 to 48 hours incubation was needed to produce results. As seen in Figure 8, the identification of bacteria using standard microbiological methods took 2-3 days or more.
Specific primers for Vibrio spp. designed from the 16s rRNA gene conservative sequence of genus Vibrio were used for LAMP detection. The LAMP assay was carried out on the suspected Vibrio isolates and ladder-like patterns were observed, which confirmed that the samples belong to the genus Vibrio (see Fig. 10). The LAMP reaction was conducted using the optimized condition of 60⁰C to 63⁰C for 60 minutes. Sybr Safe stain was added to the LAMP products and positive samples showed more intense color under UV illumination.
Figure 10. (Left) LAMP detection using six suspected Vibrio spp. isolates and (Right) Sybr Safe staining of LAMP products for Vibrio spp. detection. M-marker, V1 – V6 suspected Vibrio isolates, and (-) negative control.
The detection of Vibrio spp. is commonly performed by isolation on a selective agar medium followed by biochemical and serological testing (Harwood et al. 2004), which is arduous, time-consuming, and requires more than three days to finish. In contrast, LAMP offers a cost-effective and faster bacterial identification using primers designed using conserved regions of 16s rRNA specific to genus Vibrio (Xu et al. 2012). Both LAMP and PCR were performed on suspected bacterial isolates from Pangasinan and six isolates were confirmed as Vibrio spp. genera based on the two methods. The performance of LAMP and PCR on bacterial DNA isolates suggests that biochemical and serological testing for bacterial detection could be replaced by LAMP and PCR methods. Nonetheless, the requirement of thermal cycling equipment would discourage the use of PCR also as a suggested method for a low-cost diagnostic tool.
Figure 8. Standard micriobiological techniques for Vibrio spp. bacterial identification was performed simultaneously with molecular analysis. A – TSIA Biochemical test, B - TCBS selective media, C - API NE kit. 314
LAMP Product Visualization Two methods of Sybr Safe staining were done in the study. In Figure 6, the leftmost image was obtained when the staining was done simultaneously with the incubation. In this process, the stain was added in the LAMP reaction mixture before incubation and the results showed that positive LAMP products were indicated by the turbidity of the mixture, while the negative control was observed as a clear solution. Moreover, tubes were viewed
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Figure 11. Confirmation of positive LAMP products through staining, (leftmost –viral detection, middle – bacterial detection) and precipitate observation, (rightmost). (+) – positive control w/ WSSV DNA template, (-) – negative control w/o DNA template.
only under white light, which suggests a very practical technique for confirmation of LAMP products. The middle image in Figure 6 was from the established process of staining, which is the addition of the Sybr Safe stain after the LAMP reaction. The positive LAMP products by this process exhibited a more intense and brighter image under UV illumination compared to the negative control. Further, shown in the rightmost figure is the appearance of precipitate, which was observed in LAMP products with higher DNA yield. SUMMARY AND CONCLUSION In conclusion, the results presented in this study suggest that LAMP can be used in the Philippine setting as a practical alternative to conventional PCR and standard microbiological techniques. The temperature range of 55⁰C to 68⁰C for WSSV detection and 59⁰C to 67⁰ for Vibrio spp., and incubation periods of 60 minutes to 1 hour, were proven to be viable conditions for the LAMP assay, wherein all reactions were incubated initially at 95⁰C for 5 minutes and terminated at 80⁰C for 10 minutes. The LAMP assay was able to detect WSSV from L. vannamei collected from the four selected sites (Leyte - 75.00%, Batangas - 30.00%, Iloilo - 89.47%, and Bulacan - 43.33%), while samples collected from Laoag City, Ilocos Sur, tested negative. However, six bacterial isolates were confirmed as Vibrio spp. from the shrimps sampled from Bolinao, Pangasinan. Conventional PCR and microbiological methods were performed along with LAMP for comparison and further confirmation. WSSV detection using the LAMP assay was 10 times more sensitive than PCR, and bacterial identification of Vibrio spp. through the LAMP reaction was less time-consuming than the traditional microbiological methods. Visualization of LAMP products using Sybr Safe stain also offers an advantage, since it could eliminate the commonly used agarose gel electrophoresis which is somewhat impractical. Thus, our results suggest that a LAMP protocol is more convenient, highly sensitive, and more rapid than the established methods for disease diagnostics. Moreover, the system is cheaper and practical since it does not require an expensive PCR machine, which can benefit the small-scale shrimp industry. Vol. 7 | No. 1 | 2014
The optimum LAMP conditions that were determined and tested in this study could be readily applied, or adapted, on-site. Testing the foregoing LAMP protocols for pathogen detection on small-scale shrimp farms is a primary step in the adoption of LAMP technology in the Philippines. ACKNOWLEDGEMENTS This research was supported in part by the project, “Biotechnology for Shrimp: Utilization of Molecular Technologies to Elucidate Shrimp Immunity and Develop Disease Diagnostics”, funded by the Department of Science and Technology. Special thanks to the Thomas Aquinas Research Complex, University of Santo Tomas, for housing the project. Special thanks also to the laboratory technician Charles Gerald Ganal, and project staff members Erica M. Ocampo, Patrick Ellis Go, Ricardo S. Balog, Paul Rodrigo F. Cordero, Donna May de la Cruz-Papa, Research Assistants of the NFRDI - Genetic Fingerprinting Laboratory (GFL), and the graduate students of The UST Graduate School, Ma. Sheila de Jesus, Reuben Jerome Atayde, Ritchie Gorospe, Irma Dabu, and Vivian Villegas, who have helped in this study. CONFLICTS OF INTEREST There is no conflict of interest among authors, institutions and individuals mentioned above in the conduct of this study and the preparation and submission of this manuscript. CONTRIBUTIONS OF INDIVIDUAL AUTHORS Dr. Mary Beth B. Maningas conceived and designed the experiments. Amalea Dulcene D. Nicolasora performed the experiments. Both worked together on the data analysis and writing of the manuscript. Dr. Maningas and Benedict A. Maralit designed and developed the primers. Dr. Mudjekeewis D. Santos and Dr. Christopher Marlowe A. Caipang provided technical inputs as well as laboratory resources, and Dr. Adelaida Calpe served as a consultant for the project.
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Philippine Science Letters
Vol. 7 | No. 1 | 2014
