Background

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 [1]. For susceptible young sheep in an epidemic situation, mortality canreach 90% [2]. 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 [10]. 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 [11]. In addition, LAMP products can be detected by direct fluorescence [12]. Other fluorescent dyes, such as ethidium bromide, SYBR green andEvagreen, have also been used for visualization of LAMP products [13]. 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 [14]. 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.

Results

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).

Figure 1
figure 1

Analysis of LAMP products. LAMP amplification products wereanalyzed using agarose gel electrophoresis (A) and visuallyinspected with SYBR green I dye under daylight conditions against ablack background (B). M: DNA marker; 1: Plasmid containing theF1L gene; 2: Genomic DNA of ORFV; 3: DNA from healthy goats; 4:Water.

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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).

Figure 2
figure 2

Sensitivity of LAMP. Agarose gel electrophoresis (A) andvisual inspection using SYBR Green I staining of the LAMP products(B). M: DNA marker; 1–9 are the reaction results from a10-fold serial dilution of plasmid containing the F1L gene from108 to 100 copies per reaction; 10: Negativecontrol.

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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).

Figure 3
figure 3

Specificity of LAMP. Agarose gel electrophoresis (A) andvisual inspection using SYBR Green I staining (B). M: DNA marker;1: Plasmid containing the F1L gene; 2: Negative control; 3–12:ORFV/HB/CHA, ORFV/Vaccine/CHA, ORFV/Xinjiang/CHA, ORFV/Chongqing/CHA,ORFV/Shanxi/CHA, ORFV/Guangxi/CHA, ORFV/Gansu/CHA, ORFV/Liaoning/CHA,ORFV/Jilin/CHA and ORFV/Sichuan/CHA; 13 and 14: FMDV/O/CHA and FMDV/AsiaI/JS; 15 and 16: Capripox virus/China Vaccine and Capripoxvirus/Henan/CHA.

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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).

Figure 4
figure 4

Sensitivity and specificity of real-time PCR assay. The standardcurve (A) and dissociation curve (B) were generated usingknown concentration of recombinant plasmid containing the B2L gene from108 to 100 copies. The dissociation curves ofreal-time PCR used to detect ORFV and other select viruses(C).

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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.

Table 1 Comparison of LAMP and real-time PCR for detection of orf fromintracutaneous and cohabitation infections
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Discussion

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 [11]. 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 [8]. 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.

Conclusions

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 [22] 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).

DNA extraction

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.

LAMP assay

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. [24], 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.

Table 2 Sequence of primers designed for LAMP amplification of the F1L geneof ORFV
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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. [25]. 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.

Real-time PCR

The primers for SYBR Green I-based real-time PCR were synthesized according tothe published reference [7], 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.