Rapid and Sensitive Detection of Singapore grouper iridovirus by Loop-Mediated Isothermal Amplification【精品论文大全】 .doc

精品论文推荐Rapid and Sensitive Detection of Singapore grouper iridovirus by Loop-Mediated Isothermal AmplificationMao Xinliang 1，Zhou Sheng1，Xu Dan，Gong Jie，Cui Huachun，Qin Qiwei *State Key Laboratory of Biocontrol，College of Life Science，Sun Yat-sen University，Guangzhou（510275）E-mail：lssqinqwmail.sysu.edu.cnAbstractAims: The aim of this paper is to develop a loop-mediated isothermal amplification (LAMP) methodfor rapid, sensitive and inexpensive detection of SGIV in grouper, Epinephelus sp.Methods and Results: A set of six specific primers was designed by targeting the SGIV ORF-014L. With Bst DNA polymerase large fragment, the target DNA can be amplified as early as 20 min at 65°C in a simple water bath. The detection limit is about 0.02 fg (equivalent to 6.3 copies) of plasmid ORF-014L. LAMP products could be judged with three different methods. There were no cross reactions with 7 other aquatic animal viruses indicating high specificity of the LAMP. The LAMP method was applied to detect SGIV in virus infected GP cells and grouper tissues effectively. Conclusions: The LAMP described in this study is a cheap, sensitive, specific and rapid protocol for detection of SGIV in cells and in grouper tissues.Significance and Impact of the Study: The developed LAMP method can be simply applied both in field condition and in laboratory operation for specific detection of SGIV infection.Keywords：SGIV；LAMP；Grouper；Nested PCR.1. IntroductionIridoviruses are icosahedral deoxyriboviruses with a large double-stranded DNA genome that can be categorized into five genera (Williams et al., 2005). In recent years, iridoviruses are well known as causative agents of serious systemic diseases among feral, cultured food and ornamental fish, and have been identified from more than 100 fish species in the last decade worldwide (Piaskoski and Plumb,1999; Hyatt et al., 2000; Wang et al., 2007). These viruses have attracted much attention because of their ecological impacts and caused tremendous economic losses in the aquaculture industry in various parts of the world (Langdon et al., 1986; Ahne et al., 1989; Inouye et al., 1992; Pozet et al., 1992; Bloch and Larsen, 1993; Kasornchandra and Khongpradit, 1995; Plumb et al., 1996; Tapiovaara et al.,1998; Qin et al., 2003). Grouper, Epinephelus spp., the major species being maricultured in China and Southeast Asian countries, are high priced and popular seafood fish. In recent years, with the rapidly developing marine farming activities, outbreaks of iridoviral diseases have affected severely many highly valued species such as grouper causing heavy economic losses (Chua et al., 1994; Chou et al.,1998). Recently, a pathogenic iridovirus was isolated from diseased brown-spotted grouper, Epinephelus tauvina and Malabar grouper, Epinephelus malabaricus in our laboratory using the method of cell culture isolation. The virus caused more than 90% mortality in cultured grouper, and could induce an advanced cytopathic effect (CPE) in a grouper cell line (GP). The diseased fish spleens enlarged with hemorrhage, and multifocal areas of splenic degeneration were observed. Larege round basophilic cells with displaced nuclei were detected predominantly in spleen and kidney (Qin et al.,2002). The virus has been characterised as a new species of the genus Ranairus, family Iridoiridae based on morphological, biochemical and genetic properties and named as Singapore grouper iridovirus (SGIV) (Qin et al., 2001, 2003).Several detection methods have been developed to detect the presence of SGIV in cell culture and in the cultured grouper ranging from electron microscopic observations (Qin et al., 2001),1These authors contribute equally to this work.- 13 -immunofluorescence antibody test (IFAT) and antigen-capture enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies (Qin et al., 2002; Shi et al., 2003), in situ hybridization (Huang et al., 2004), flow cytometry (FCM) (Qin et al., 2005) and a bead-based microfluidic device (Liu et al., 2005). All these methods are effective and accurate in detecting the virus infection in laboratory. However, these methods have some intrinsic disadvantages such as the requirement for expensive equipments and reagents, or laborious and time consuming, rendering their unfavorable for use on a wide-scale basis. Detection method not only rapid and sensitive but also simple and economical to handle is needed for practical application.To meet these requirements, a loop-mediated isothermal amplification (LAMP) reaction was developed as an alternative method. LAMP is a specific nucleic acid amplification method, which is easy to perform and can amplify nucleic acid at isothermal conditions at 6065 °C within 1 h incubation period (Notomi et al., 2000; Mori et al., 2001; Nagamine et al., 2002). The LAMP reaction requires four or six primers, which are based on six or eight distinct regions of the target DNA, hence it allows for a high degree of specificity for viral detection. At the end of the reaction, the presence or absence of the target DNA is judged visually by the appearance of a white precipitate of magnesium pyrophosphate or green color of the solution stained by SYBR green I. The presence of multiple bands of the LAMP reaction products in agarose gel electrophoresis indicative of a mixture composed of stem-loop DNAs with various sizes of stem and cauliflower-like structures with multiple loops induced by annealing between alternatively inverted repeats of the target sequence in the same strand (Notomi et al., 2000; Nagamine, et al., 2002). LAMP has been applied for specific detection of aquatic animal viruses such as white spot syndrome virus (WSSV) (Tomoya et al., 2004), red sea bream iridovirus (RSIV) (Christopher et al.,2004), koi herpes virus (KHV) (Hatem and Mansour, 2005) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) (Sun et al., 2006). In this study, the LAMP for specific, rapid and sensitive detection of SGIV in infected cell cultures and fish was developed.2. Materials and methods2.1 Cell cultures and virus infectionThe GP cells, a permanent marine fish cell line, derived from embryos of grouper, E. tauvina (Chew-Lim et al., 1994), were cultured in 75 cm2 flasks at 25 °C in Eagles minimum essential medium (EMEM)(Sigma,St.Louis,MO)supplementedwith5mMN-2-hydroxyethyl piperazine-N-2-ethanesulfornc acid (HEPES) buffer (Sigma), 100 IU ml1 penicillin, 100 µg ml1 streptomycin, 0.116 M sodium chloride, 7% sodium bicarbonate, and 10% fetal calf serum (Sigma). The iridovirus used in this study is SGIV, strain of A3/12/98 PPD isolated from diseased groupers, Epinephelus tauvina and E. malabaricus in Singapore (Qin et al., 2003). The GP cell culture monolayers were infected with virus at a multiplicity of infection (MOI) of approximately 0.1. When advanced cytopathic effect (CPE) was appeared, the virus-infected cells were harvested and stored at-20 °C for DNA extraction.For artificially infection, grouper in average weight of 60g were maintained in 500 L tanks (at 25 °C)filled with air-pumped circulating sea water. Each fish was injected intraperitoneally with 100µl 105TCID50 ml1 of SGIV. The organs (spleen, kidney, liver, intestines and brain) were sampled from diseased fish about one week after virus challenge.2.2 Extraction of DNADNA was prepared using both a phenol-chloroform extraction method and a tissue boiling method. Briefly, SGIV-infected fish tissue samples were homogenized with TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 8.0) and proteinase K was added to a final concentration of 50 µg ml1. The mixture was incubated for 1h at 37 °C, after which the DNA was extracted using phenol-chloroform-isoamyl alcohol (PCI) solution and precipitated with ethanol. Virus-infected cell cultures were havested andsubjected to phenol-chloroform DNA extraction directly. The DNA pellet was dissolved in TE buffer and stored at -20 °C until use. The concentration of total DNA in sample was estimated using spectrophotometer.To obtain crude DNA by tissue boiling method, approximately 100 mg of tissue from an infected fish was homogenized in 1000 µl of 2% SDS in 100 mM TrisHCl (pH 8.0), boiled for 10 min, and centrifuged at 10,000 g for 5min. The supernatant was transferred to a new tube and used immediately.2.3 Design of LAMP primersTable 1. The primers used for LAMPPrimerTypeLengthSequenceF3Forward outer20-nt5-GCACGAGTATACGGCCTCTG-3 B3Backward outer18-nt5-CGGCTACCAGCATCCAAT-3FIPForward inner(F1c+TTTT+F2) BIPBackward inner(B1c+TTTT+B2)45-mer (F1c:21-nt, F2:20-nt)45-mer (B1c:22-nt,B2:19-nt)5-GACTACGGGTTCGATGGCTGC-TT TT-AGACGATTGCGCATGAAACA-35-ACGGGAGGAAACTTGTGCTGAT-TTTT-ATGGCCCGTAGAAGTCAGT-3LFLoop Forward19-nt5-TCGCGGTTCGGGTCATCAT-3 LBLoop Backward20-nt5-TGCTGGAACGTGCAAAACCG-3A set of six species-specific LAMP primers was designed to target SGIV ORF-014L sequence (GenBank Accession No. AAS18029) as described by Notomi et al. (2000) and Yeh et al. (2005). Briefly, we first searched the nucleic acid sequences of SGIV (GenBank accession no. AY521625) using BLASTN program (Altschul et al. 1997) to choose the species-specific gene sequence suitable for diagnosis of SGIV. The SGIV ORF-014L including a digested site by the restriction enzyme Mst I was selected and used for LAMP target. The LAMP primers were designed by the PrimerExplorer V3 software program (http:/primerexplorer.jp) as following: Forward outer primer (F3), Backward outer (B3), Forward inner primer (FIP), Backward inner (BIP), Loop Forward (LF) and Loop Backward (LB). The primers sequences and the locations were indicated in Table 1 and Fig. 1.ABFig.1. Schematic diagram of primers sequences and positions for LAMP. (A). Nucleotide sequence of partial SGIV ORF-014L used to design inner and outer primers for LAMP. The nucleotide sequences and the positions used to design the primers and digest with MstI are represented by dashed lines and labels. (B). A schematic diagram showing the positions at which the primers attach for amplification of the target gene.2.4 Construction of plasmid pT-014LA recombinant plasmid (pT-014L) was constructed as template for development of the SGIV LAMP protocol. A 233 bp of SGIV ORF-014L DNA segment was amplified and cloned into pMD18-T Easy vector (Takara Bio Dalian Co. Ltd.) according to the manufacturers instructions. The DNA segment spanned the sequences between the F3 and B3 primers. The plasmid (pT-014L) constructed was used to make standard dilutions for the evaluation of the detection limit of the LAMP protocol.2.5 LAMP reactionThe LAMP was carried out in a total 25 µl reaction volume containing 0.2 µM each of F3 and B3, 0.8µM each of FIP and BIP, 0.4 µM each of the LF and LB primers, 1.0 mM dNTPs, 1M betaine (Sigma),20 mM TrisHCl (pH8.8), 10 mM KCl, 10 mM (NH4)2SO4 ,4 mM MgSO4 , 0.1% Triton X-100, 8 units of the Bst DNA polymerase large fragment (New England Biolabs), appropriate amount of template DNA. The reaction time was optimized by incubatting the mixture for 10, 20, 30, 40, 50 and 60 min at pre-determined temperature (65 °C), while the reaction temperature was optimized by incubatting the mixture at 60, 61, 62, 63, 64 and 65 °C for pre-determined time (60min). The reaction was terminated by heated at 80 °C for 5 min. The LAMP products (3 µl) were electrophoresed on 2% agarose gels and stained with ethidium bromide to determine the optimal conditions.2.6 Observation of LAMP products by naked eyesLAMP amplicons in the reaction tube were directly detected with the naked eye by adding 1.0 µl of1000-fold diluted original SYBR Green I (Molecular Probes Inc.) to the tube, and color of the solution was observed. The solution changed from light orange to green in the presence of LAMP amplicons, while it remained light orange with no amplification. Prior to the addition of SYBR Green I, white turbidity of the reaction mixture by magnesium pyrophosphate (by-product of LAMP) was also inspected. Reaction mixture (3 µl) was analysed by 2 % agarose gel electrophoretic, ethidium bromide stained and visualized.2.7 Nested PCR detectionTwo pairs of PCR primers were designed for nested PCR. For the first step PCR, the outer primer set (F-014L: 5'-TACGGGCACGCACGAATA-3' and R-014L 5'-CGGCTACCAGCATCCAA-3') was used to amplify a 233 bp of the virus ORF-014L segment. The PCR reactions contained 4 µl dNTP( 2.5 mM ), 5 µl 10 × buffer with MgCl2 (25 mM), 2 µl of both F-014L and R-014L primers ( 10 µM ),1 µl of DNA sample, 0.5 µl rTaq (5U µl-1) and distilled water in a final volume of 50 µl. Amplification conditions were as follows: 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 45 s and followed by a final elongation at 72 °C for 5 min. For the second step PCR, the inner primer set (IF-014L: 5'-CGCATGAAACATAAACG-3' and IR-014 (same as to R-014L):5'-CGGCTACCAGCATCCAA-3') targeted to the first step PCR product amplified a 192 bp of fragment using the same conditions as stated above. The amplified DNA fragments were analyzed by electrophoresis on a 1 % ethidium bromide-stained agarose gel in Tris-boric acid-EDTA (TBE) buffer (pH 8.0).2.8 Specificity of LAMP assayThe specificity of the assay was tested by using templates from SGIV and 7 other aquatic animal viruses including 3 iridoviruses of tiger frog virus (TFV)，soft-shelled turtle iridovirus (STIV) and lymphocystis disease virus (LCDV), and 4 another unrelated fish viruses of koi herpes virus (KHV),nervous necrosis virus (NNV), infectious haematopoietic necrosis virus (IHNV) and spring viremia of carp virus (SVCV). In order to confirm specificity of the LAMP, the LAMP products were digested with the restriction enzyme Mst I (Takara Bio Dalian Co. Ltd.) to be cut the F1-loop. Nested PCR was carried out as control assay.2.9 Sensitivity of the LAMP assayThe detection limits of the SGIV LAMP assay were evaluated using 10-fold serial dilutions of plasmid (pT-014L). The plasmid DNA (0.2 µg µl-1 equivalent to 6.3×1010copies/µl) was 10-fold serially diluted and 1 µl of each dilution was used as templates for LAMP reaction. The reaction was performed at65°C for 60 min, and compared with nested PCR assay.2.10Application of LAMP to detect virus infection in GP cells and in fish tissuesIn order to evaluate the optimal LAMP assays for detection of SGIV, total DNA was extracted from SGIV infected GP cells at different time of post infection (1 to 48 h) by phenol-chloroform method, and from virus-challen