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Comparison of a loop-mediated isothermal amplification for orf virus withquantitative real-time PCR
Virology Journalvolume 10, Article number: 138 (2013)
Orf virus (ORFV) causes orf (also known as contagious ecthyma or contagiouspapular dermatitis), a severe infectious skin disease in goats, sheep andother ruminants. Therefore, a rapid, highly specific and accurate method forthe diagnosis of ORFV infections is essential to ensure that the appropriatetreatments are administered and to reduce economic losses.
A loop-mediated isothermal amplification (LAMP) assay based on theidentification of the F1L gene was developed for the specific detection ofORFV infections. The sensitivity and specificity of the LAMP assay wereevaluated, and the effectiveness of this method was compared with that ofreal-time PCR.
The sensitivity of this assay was determined to be 10 copies of a standardplasmid. Furthermore, no cross-reactivity was found with either capripoxvirus or FMDV. The LAMP and real-time PCR assays were both able to detectintracutaneous- and cohabitation-infection samples, with a concordance of97.83%. LAMP demonstrated a sensitivity of 89.13%.
The LAMP assay is a highly efficient and practical method for detecting ORFVinfection. This LAMP method shows great potential for monitoring theprevalence of orf, and it could prove to be a powerful supplemental tool forcurrent diagnostic methods.
The orf virus (ORFV) is the prototype member of the Parapoxvirus genus within thePoxviridae family. The ORFV has a worldwide distribution and causes an infectiousskin disease known as contagious ecthyma in goats, sheep and other ruminants . For susceptible young sheep in an epidemic situation, mortality canreach 90% . Therefore, a practical and reliable method for the diagnosis of ORFVinfections is required.
For diagnosis of such infections, clinical signs, virus isolation and electronmicroscopy are commonly used along with serological tests. However, these methodsare laborious, time-consuming and, in some cases, not effective. For example, virusisolation can be unsuccessful at times, even when virus-like particles are observedin the lesions resulting from infection. Goats and sheep are commonly infected withthe virus, yet serological tests have not confirmed the cause of clinical signs.PCR-based diagnostic assays have been developed for the sensitive and specificdetection of ORFV infections [3–7]. However, these assays have not been widely adopted in resource-poorregions due to their relatively complex nature, as well as the need for bothexpensive equipment and highly trained personnel [8, 9].
Loop-mediated isothermal amplification (LAMP) is a simple technique that rapidlyamplifies specific DNA sequences with high sensitivity under isothermal conditions . LAMP products can easily be detected by the naked eye due to theformation of magnesium pyrophosphate, a turbid white by-product of DNA amplificationthat accumulates as the reaction progresses . In addition, LAMP products can be detected by direct fluorescence . Other fluorescent dyes, such as ethidium bromide, SYBR green andEvagreen, have also been used for visualization of LAMP products . Furthermore, Thekisoe et al. have reported that LAMP reagents arerelatively stable even when stored at 25 or 37°C, which supports the use ofLAMP in field conditions and resource-poor laboratories . Recently, LAMP assays targeting the B2L and DNA polymerase genes of ORFVhave been developed, and these methods were found to be powerful diagnostic tools [8, 9].
The F1L gene is part of the highly conserved central region of the ORFV genome [15, 16]. In the present study, a LAMP assay was developed to specificallyidentify the F1L gene sequence to facilitate the detection of ORFV infections andthe effectiveness of this method was compared with that of real-time PCR.
Detection of LAMP product
The LAMP products were electrophoresed on a 1.5% agarose gel stained withethidium bromide solution and visualized under UV light. In addition, visualinspection of the LAMP products was performed by adding SYBR Green I to thereaction mixture tube and observing the fluorescent signals of the solutionsunder daylight conditions against a black background (Figure 1).
Sensitivity of LAMP
To determine the detection limit of the LAMP assay, 10-fold serial dilutions ofthe PF1L were amplified using LAMP. As determined using both 1.5% agarose gelelectrophoresis and color inspection with SYBR Green I dye, the detection limitof the LAMP assay was determined to be 10 copies of DNA (Figure 2).
Specificity of LAMP
The specificity of the LAMP assay was evaluated using the genomic DNA of 10 knownORFV isolates, capripox virus and FMDV. Only the specific ORFV target DNA wasamplified by LAMP. No cross-reactivity was observed with the DNA samples ofcapripox virus or FMDV. These results confirm that the LAMP assay is highlyspecific (Figure 3).
Sensitivity and specificity of real-time PCR
To determine the detection limit of the real-time PCR, the copy number of arecombinant plasmid containing the B2L gene (PB2L), which was previouslyconstructed by our lab, was calculated as described below. PB2L was amplifiedfrom 10-fold serial dilutions using real-time PCR. The detection limit was foundto be 10 copies/μl. The real-time PCR slope was −3.218, with anR2 = 1 and a reaction efficiency of 104.5%. Moreover,the standard curve generated using the 10-fold serial dilutions of the plasmidwas linear over eight orders of magnitude (1 to 108 copies/μl),demonstrating that real-time PCR can be used to accurately quantify this targetDNA over a large range of concentrations (Figure 4A).
The primers chosen for real-time PCR were initially validated by monitoringproduct amplification with SYBR Green I. Melting curve analysis showed a uniquepeak at 86°C, indicating the formation of a single PCR product without thepresence of nonspecific amplification products or primer dimers(Figure 4B). To further determine the specificityof the amplification reaction with the chosen primers, the SYBR Green I-basedreal-time PCR was performed using DNA from 10 different isolates of ORFV,capripox virus and FMDV. All ORFV DNA samples tested using SYBR Green I-basedreal-time PCR yielded a positive result. However, the DNA samples from capripoxvirus and FMDV did not yield an amplification signals (Figure 4C).
Evaluation of the LAMP assay using samples from experimentally infectedgoats
To evaluate the practicality and efficiency of the LAMP assay, its ability todetect ORFV infections was tested using the skin lesions from experimentallyinfected goats. Forty-six samples of skin lesions from four experimentallyinfected goats were tested using the LAMP (by both visual inspection and agarosegel electrophoresis methods) and real-time PCR assays. Forty-one samples weredetermined to be positive by both LAMP and real-time PCR. One sample wasdetermined to be negative by LAMP assay but positive by real-time PCR. Thesensitivities of the LAMP and real-time PCR assays were 89.13% and 91.30%,respectively (Table 1), and the results were verysimilar between the two assays.
Orf is distributed throughout many countries [2, 17–23], and ORFV infections can cause weight loss and poor development as thedisease prevents host animals from feeding. Therefore, the development of a rapid,simple and sensitive detection method for ORFV infections is required. For thediagnosis of ORFV infections, the LAMP assay has many advantages, such assimplicity, rapidity and inexpensiveness, compared with other nucleic acid-basedtests [8, 9]. In addition, previous reports have demonstrated that the sensitivity ofthe LAMP is higher than that of conventional PCR or nested PCR for detecting ORFVinfections [8, 9]. Therefore, this method shows great potential for use in resource-limitedveterinary laboratories in developing countries (e.g., China), where many endemicdiseases exist. In the present study, we evaluated the sensitivity and specificityof the rapid diagnostic LAMP method.
The simplest way of detecting LAMP products is to visually inspect the whiteturbidity that results from magnesium pyrophosphate accumulation as a by-product ofthe reaction . However, a small amount of this white precipitate is not alwaysdistinguishable from other white precipitates, such as proteins or carbohydratesthat can be derived from the templates. Therefore, existing“field-friendly” LAMP-based detection systems are still imperfect.Previous report has demonstrated that amplified DNA can be stained (using PicoGreenor ethidium bromide) and visualized in solution with the same sensitivity as agarosegel electrophoresis . In this study, we used the dye SYBR Green I to detect the amplified DNAproducts. Positive and negative reactions could be differentiated by distinctlydifferent colors when viewed under daylight conditions against a black background(Figure 1). Consistent with previous report, thiscolor inspection method was found to have the same detection limit as agarose gelelectrophoresis (Figure 2), which should facilitate therapid screening of samples without the need for gel electrophoresis. These resultsindicate that visual color inspection of LAMP products using the SYBR Green I dyeshould be practical under field conditions.
The reliability of a LAMP assay depends largely on the specificity of the primer setsbeing used. These primer sets in this study—two outer primers (F3 and B3) andtwo inner primers (FIP and BIP)—allow for the recognition of six sites in thetarget sequence that are specific to the F1L gene and that are necessary for theLAMP reaction to occur. Based on the sequences of the ORFV F1L gene available inGenBank and the sequences of ORFV isolates from China, we designed and testedseveral sets of primers using comparative experiments, and the primer sets thatyielded the highest specificity and sensitivity in the LAMP assay is reported here.Our LAMP assay showed high specificity and sensitivity, as it yielded positiveresults for all 10 of the known ORFV isolates but did not amplify the negativecontrol samples (Figure 3).
Samples from experimentally infected goats were tested using both the LAMP andreal-time PCR assays. The LAMP assay correctly identified 41 out of 46 samples aspositive (89.13%), and the real-time PCR assay correctly identified 42 out of 46samples as positive (91.30%). ORFV DNA could be detected in most of theintracutaneous- and cohabitation-infection samples from the time of lesionpresentation to recovery (Table 1). Therefore, weobserved a very good agreement between the LAMP and real-time PCR results. Only onesample was identified as negative by LAMP (both by visual inspection and agarose gelelectrophoresis) but as positive by real-time PCR. This discrepancy was most likelydue to very low ORFV levels in this sample, which may have been beyond the detectionlimit of LAMP. This result suggests that the sensitivity of LAMP is slightly lowerthan that of real-time PCR. However, it is important to consider that real-time PCRis a time-consuming procedure that requires expensive and relatively complicatedequipment, including a thermal cycler with real-time monitoring and data-analysissystems. Therefore, this LAMP-based assay has clear advantages over real-time PCR interms of practicality, and it can be easily used in any standard diagnosticlaboratory, particularly in developing countries where the disease is prevalent.
We show that a LAMP assay based on amplification of the ORFV F1L gene is rapid,highly specific and accurate for the detection of ORFV infections. The sensitivityof this LAMP (89.13%) was higher than previously reported (70% and 74.3%,respectively). Furthermore, this color inspection method with SYBR Green I dye hadthe same sensitivity as agarose gel electrophoresis, which should facilitate therapid large-scale screening of samples and the rapid diagnosis of ORFV infectionsunder field conditions without the use of agarose gel electrophoresis. Therefore, weconclude that this LAMP is a practical and reliable method for detecting ORFVinfections, and we suggest that this test can be adopted as a powerful supplementaltool to current diagnostic assays.
Materials and methods
Intracutaneous and cohabitation infection of goats
Four healthy 12- to 14-week-old goats were selected for this study. The ORFVstrain ORFV/HB/09  was propagated in bovine testicular cells using Eagle’s minimalessential medium (Shanghai Gaochuang Medical Science And Technology Co., Ltd,Shanghai, China) containing 10% fetal calf serum(TCID50 = 10-5.3/0.1 ml), which was usedto infect the goats to prepare the ORFV-positive samples. Two goats wereinoculated intradermally on oral mucosa with viral supernatant (0.2 ml per goat)and housed individually. After 7 days, each non-inoculated goat was housed withone inoculated goat, thereby making two replicate cohabitation groups. Theclinical signs and macroscopic lesions of goats infected via both routes wereobserved. The samples of the skin lesions around the muzzle and lips werecollected from the time of lesion presentation to recovery, and these sampleswere used to evaluate of the LAMP method. All experimental procedures and animalcare were conducted in accordance with the guidelines and the regulations of theGansu Animal Care and Use Committee. The experimental protocol was approved bythe Ethical Committee of the Lanzhou Veterinary Research Institute, ChineseAcademy of Agricultural Sciences (XYXK- (Gan) 2010–003).
Briefly, the tissue sample (25 mg) was mechanically homogenized in 250 μl ofphosphate-buffered saline (PBS) in a tube using a pellet pestle device. Thehomogenates were centrifuged at 2000 ×g for 3 min, and thesupernatant was collected. The DNA templates for the LAMP and RT-PCR assays wereextracted using the Universal Genomic DNA Extraction kit (TaKaRa BiotechnologyCo., Ltd, Dalian, China) according to the manufacturer’s protocol. DNAsamples extracted from healthy goats were used in parallel with the experimentalsamples as negative controls.
The LAMP primer sets (Table 2) were designed from theF1L gene region of the published sequence of ORFV isolate Jilin-Nongan (GenBankaccession no. JQ271535.1) using the Primer Explorer V4 Software(http://primerexplorer.jp/elamp4.0.0/index.html). The LAMPreaction was carried out as previously described by Thekisoe et al. , with minor modifications. The LAMP reaction mixture with a totalvolume of 25 μl contained: 12.5 μl of 2 × LAMP reaction buffer(40 mM Tris–HCl [pH 8.8], 20 mM KCl, 16 mM MgSO4, 20 mM(NH4)2SO4, 0.2% Tween 20, 1.6 M Betaine,2.8 mM of each dNTP), 1 μl (8 units) of Bst DNA polymerase (New EnglandBiolabs, Massachusetts, USA), 2.6 μl primer mix (forward inner primer (FIP)and backward inner primer (BIP) at 40 pmol each, forward outer primer (F3) andforward outer primer (B3) at 10 pmol each), 3 μl of template DNA and 5.9μl of double distilled water. LAMP was performed at 63°C in a waterbath for 1 hour, and samples were identified as positive by agarose gelelectrophoresis. LAMP reactions were also stained with SYBR green I dye (TaKaRaBiotechnology Co., Ltd, Dalian, China), and samples were identified as positiveby a specific color change when viewed under daylight conditions against a blackbackground.
Sensitivity and specificity of the LAMP assay
The copy number of the recombinant plasmid containing the F1L gene (PF1L), whichwas constructed previously by our lab, was calculated as described by Wang etal. . Briefly, the concentration of PF1L (X) was determined byspectrophotometry and converted to number of molecules using the followingformula:copies/ml = 6.02 × 1023 × X/Y(X = OD260 × 50 × 10-6g/ml × dilution factor; Y = the length of PF1L(bp) × 660). The sensitivity of the LAMP assay was thendetermined using the 10-fold serial dilutions of PF1L. Additionally, thespecificity of the LAMP assay was determined using DNA from 10 known isolates ofORFV, 2 strains of capripox virus and 2 strains of FMDV.
The primers for SYBR Green I-based real-time PCR were synthesized according tothe published reference , 5′-CAGCAGAGCCGCGTGAA-3′, and5′-CATGAACCGCTACAACACCTTCT-3′. Real-time PCR was performed with thedetection of the B2L gene of ORFV on an ABI PRISM 7500 thermocycler. Thereal-time PCR reactions were prepared for a 25 μl reaction volumecontaining 2 × SYBR Premix Ex Taq II supplemented with ROX II,the primers (10 μM each) and 3 μl of DNA template. The cyclingparameters were as follows: preheat at 95°C for 30 s, then 40 cycles of95°C for 5 s and 64°C for 20 s. After amplification, the data werethen analyzed using the 7500 System software. A melting curve analysis wasperformed to verify the uniqueness of the amplified product by its specificmelting temperature.
Haig DM, Mercer AA: Ovine Diseases: Orf. Vet Res 1998, 29: 311-326.
Mondal B, Bera AK, Hosamani M, Tembhurne PA, Bandyopadhyay SK: Detection of orf virus from an outbreak in goats and its genetic relationwith other parapoxviruses. Vet Res Commun 2006, 30: 531-539. 10.1007/s11259-006-3270-z
Inoshima Y, Morooka A, Sentsui H: Detection and diagnosis of parapoxvirus by the polymerase chain reaction. J Virol Methods 2000, 84: 201-208. 10.1016/S0166-0934(99)00144-5
Torfason EG, Gunadottir S: Polymerase chain reaction for laboratory diagnosis of orf virusinfections. J Clin Virol 2002, 24: 79-84. 10.1016/S1386-6532(01)00232-3
Kottaridi C, Nomikou K, Lelli R, Markoulatos P, Mangana O: Laboratory diagnosis of contagious ecthyma: comparison of different PCRprotocols with virus isolation in cell culture. J Virol Methods 2006, 134: 119-124. 10.1016/j.jviromet.2005.12.005
Chan KW, Hsu WL, Wang CY, Yang CH, Lin FY, Chulakasian S, Wong ML: Differential diagnosis of orf viruses by a single-step PCR. J Virol Methods 2009, 160: 85-89. 10.1016/j.jviromet.2009.04.025
Gallina L, Dal Pozzo F, McInnes CJ, Cardeti G, Guercio A, Battilani M, Ciulli S, Scagliarini A: A real time PCR assay for the detection and quantification of orf virus. J Virol Methods 2006, 134: 140-145. 10.1016/j.jviromet.2005.12.014
Tsai SM, Chana KW, Hsu WL, Chang TJ, Wong ML, Wang CY: Development of a loop-mediated isothermal amplification for rapid detectionof orf virus. J Virol Methods 2009, 157: 200-204. 10.1016/j.jviromet.2009.01.003
Li J, Song D, He W, Bao Y, Lu R, Su G, Wang G, Lu H, Zhao K, Gao F: Rapid detection of orf virus by loop-mediated isothermal amplification basedon the DNA polymerase gene. Arch Virol in press 10.1007/s00705-012-1526-1
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T: Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000, 28: e63. 10.1093/nar/28.12.e63
Mori Y, Nagamine K, Tomita N, Notomi T: Detection of loop-mediated isothermal reaction by turbidity derived frommagnesium pyrophosphate formation. Biochem Biophys Res Commun 2001, 289: 150-154. 10.1006/bbrc.2001.5921
Tomita N, Mori Y, Kanda H, Notomi T: Loop-mediated isothermal amplification (LAMP) of gene sequences and simplevisual detection of products. Nat Protoc 2008, 3: 877-882. 10.1038/nprot.2008.57
Qiao YM, Guo YC, Zhang XE, Zhou YF, Zhang ZP, Wei HP, Yang RF, Wang DB: Loop-mediated isothermal amplification for rapid detection of Bacillusanthracis spores. Biotechnol Lett 2007, 29: 1939-1946. 10.1007/s10529-007-9472-9
Thekisoe OMM, Bazie RSB, Coronel-Servian AM, Sugimoto C, Kawazu S, Inoue N: Stability of loop-mediated isothermal amplification (LAMP) reagents and itsamplification efficiency on crude trypanosome DNA templates. J Vet Med Sci 2009, 71: 471-475. 10.1292/jvms.71.471
Mercer AA, Fleming S, Robinson A, Nettleton P, Reid H: Molecular genetic analysis of parapoxviruses pathogenic for humans. Arch Virol 1997, 13: 25-34.
Scagliarini A, Ciulli S, Battilani M, Jacoboni I, Montesi F, Casadio R, Prosperi S: Characterisation of immunodominant protein encoded by the F1L gene of orfvirus strains isolated in Italy. Arch Virol 2002, 147: 1989-1995. 10.1007/s00705-002-0850-2
Inoshima Y, Murakami K, Wu D, Sentsui H: Characterization of parapoxviruses circulating among wild Japanese serows(Capricornis crispus). Microbiol Immunol 2002, 46: 583-587.
Hosamani M, Bhanuprakash V, Scagliarini A, Singh RK: Comparative sequence analysis of major envelope protein gene (B2L) of Indianorf viruses isolated from sheep and goats. Vet Microbiol 2006, 116: 317-324. 10.1016/j.vetmic.2006.04.028
Chan KW, Lin JW, Lee SH, Liao CJ, Tsai MC, Hsu WL, Wong ML, Shih HC: Identification and phylogenetic analysis of orf virus from goats inTaiwan. Virus Genes 2007, 35: 705-712. 10.1007/s11262-007-0144-6
Abrahão JS, Campos RK, Trindade GS, Guedes MIM, Lobato ZIP, Mazur C, Ferreira PCP, Bonjardim CA, Kroon EG: Detection and phylogenetic analysis of Orf virus from sheep in Brazil: a casereport. Virol J 2009, 4: 47-50.
Jonatas SA, Rafael KC, Giliane ST, Maria IMG, Zelia IPL, Carlos M, Paulo CPF, Claudio AB, Erna GK: Detection and phylogenetic analysis of orf virus from sheep in Brazil: acasereport. J Virol 2009, 6: 47. 10.1186/1743-422X-6-47
Zhang KS, Shang YJ, Jin Y, Wang GX, Zheng HX, He JJ, Lu ZX, Liu XT: Diagnosis and phylogenetic analysis of Orf virus from goats in China: a casereport. Virol J 2010, 7: 78-82. 10.1186/1743-422X-7-78
Zhao K, Song D, He W, Lu H, Zhang B, Li C, Chen K, Gao F: Identification and phylogenetic analysis of an orf virus isolated from anoutbreak in sheep in the Jilin province of China. Vet Microbiol 2010, 142: 408-415. 10.1016/j.vetmic.2009.10.006
Thekisoe OMM, Rambritch NE, Nakao R, Bazie RS, Mbati P, Namangala B, Malele I, Skilton RA, Jongejan F, Sugimoto C, Kawazu SI, Inoue N: Loop-mediated isothermal amplification (LAMP) assays for detection ofTheileria parva infections targeting the PIM and p150 genes. Int J Parasitol 2010, 40: 55-61. 10.1016/j.ijpara.2009.07.004
Wang PJ Master's thesis. In Development and application of real-time PCR for detection of Toxoplasmagondii. Chongqing University of Medical Sciences; 2005.
This investigation was financially supported by a grant from the National ModernMeat Caprine Industrial Technology System (nycytx-39). Funding agency had norole in experiment design, data collection, analysis or interpretation, nor inmanuscript writing, revision or in manuscript submission.
The authors declare that they have no competing interests.
GXW, YJS and XTL designed the experiment. GXW performed lab work. GXW and YHWparticipated in data analysis and drafted the manuscript. XTL, YJS and HT revisedthe manuscript. All the authors read and approved the final manuscript.