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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6851—Quantitative amplification

Definitions

• the present invention relates to a method for detecting viral infection in shrimps which is caused by the infectious hypodermal and haematopoietic necrosis virus (IHHNV). More particularly, the present invention provides a high-throughput real- time viral detection method for screening and identifying IHHNV in shrimps, as well as a rapid detection system and kit thereof; which are capable of controlling the prevalence of the IHHNV in shrimps, thus enhancing the breeding program in the shrimp-farming industry, especially for giant fresh water prawn, Macrobrachium rosenbergii.
• IHHNV infectious hypodermal and haematopoietic necrosis virus
• the giant freshwater prawn, M. rosenbergii is the shrimp species most used for commercial farming worldwide. This species of shrimps is known as one of the high- value aquaculture products as it is a good food source which is rich in proteins. Besides, it is also commercially important as a valuable export product.
• ill management in shrimp-farming often causes widespread of viral infections, which have also become the major limiting factor in the shrimp-farming program of M. rosenbergii.
• viral infections result in acute epizootics which frequently associated with explosive death among shrimp populations, rendering substantial economic losses in commercial aquaculture and posing threats to the continue of valuable wild stocks.
• IHHNV infection is one of the major causes of shrimp diseases, especially in M.
• PCR amplifications there are also other examples of PCR amplifications disclosed in the prior art for the detection of IHHNV or the related shrimp viruses.
• a method for detecting IHHNV in a biological sample by nested PCR technique is disclosed in Malaysian Patent application No. PI2011002235. This method comprises the steps of amplifying conserved segment of the virus in a biological sample by a first PCR using a first primer set; and contacting the amplified conserved segment with a second primer set in a second PCR.
• this nested PCR method may not be sufficiently sensitive to detect the presence of IHHNV in shrimps at its development stage.
• China Patent No. 1786711 also provides a fluorescent quantitative (FQ)-PCT method and kit for detecting IHHNV, in which a forward and a reverse primer, as well as a fluorescent probe is provided.
• This fluorescent probe uses rhodamine (TAM) as fluorophore and tetramethylrhodamine (TAMRA) as quencher.
• TAM rhodamine
• TAMRA tetramethylrhodamine
• the IHHNV is a 20 to 22 nm, icosahedral, non-enveloped virus, with a density of 1.40 g ml "1 in CsCl and contains a single- stranded linear DNA genome.
• This virus can cause severe growth retardtion, resulting in serious impediment in the production of commercial freshwater prawn-farming. It can be persistent in the healthy carrier for life and be passed onto progeny through vertical transmission and to other populations through horizontal transmission. As the wild or pond-reared brookstock may be asymptomatic individuals, they inevitably pose a biosecurity risk in shrimp-farming. Therefore, the development of IHHNV detection method in the healthy carriers of viral pathogen is desirable.
• the primary object of the present invention is to provide a method for detecting IHHNV quantitatively in real-time in a sample of shrimps, especially M. rosenbergii.
• Another object of the present invention is to provide a diagnostic kit for rapid detection of IHHNV which is based on a specifically designed real-time polymerase chain reaction (PCR) technique.
• PCR polymerase chain reaction
• Still another object of the present invention is to provide a method and a kit for detecting IHHNV which is sensitive and reliable, and able to detect the presence of IHHNV in M. rosenbergii, at its various development stages. Besides infected individuals, the healthy carriers of viral pathogens shall also be detected.
• Yet another object of the present invention is to provide a system or a kit which is capable of accurately and rapidly detecting the presence of EHHNV in shrimps, thus facilitating the monitoring system of IHHNV infection and preventing viral disease outbreak that can potentially cause severe economic loss in the country.
• Further object of the present invention is to develop a system which effectively controls the prevalence of aquatic viral diseases in shrimps, especially IHHNV infections, thus enhancing the productivity and breeding program of the shrimp- farming industry.
• Another further object of the present invention is to produce high quality domesticated potential broodstocks which is safe for the environment, and high quality aquatic foods which are safe to be consumed.
• one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention describes a method for quantitatively detecting IHHNV in a biological sample, comprising the steps of obtaining a primer pair having nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, and a fluorescent probe having a nucleotide sequence set forth in SEQ ID NO: 3; amplifying a non-structural protein coding region of the virus in the biological sample by a real-time PCR using the primer pair and the fluorescent probe; and determining the presence and quantity of the virus in the biological sample with reference to a positive control plasmid.
• the biological sample is a DNA or cDNA template.
• This biological sample is preferably derived from tissues or whole bodies of an aquatic animal collected at different development stages from different growth environments.
• the aquatic animal is a shrimp species of M. resenbergii; and the different development stages preferably include adult, juvenile and larval stages.
• the primer pair and the fluorescent probe are designed from a non-structural protein coding region of the IHHNV genome.
• the fluorescent probe is synthesized with a reporter fluorescent dye at its 5' end and a non- fluorescent quencher at its 3' end.
• the reporter fluorescent dye is 5- carboxyfluorescein (RAM); whereas the non-fluorescent quencher is minor groove binder (MGB).
• MGB minor groove binder
• Further embodiment of the present invention is a primer pair for use in a real-time PCR-based quantitative detection of IHHNV in a biological sample, comprising nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2.
• Another further embodiment of the present invention is a fluorescent probe for use in a real-time PCR- based quantitative detection of IHHNV in a biological sample, comprising a nucleotide sequence set forth in SEQ ID NO: 3.
• the primer pair and the fluorescent probe are preferably designed to specifically recognize and amplify a conserved open reading frame (ORF1) of a nonstructural protein coding region in the IHHNV genome.
• ORF1 conserved open reading frame
• the fluorescent probe is preferably having a reporter fluorescent FAM dye at its 5' end and a non-fluorescent MGB quencher at its 3' end.
• Still another further embodiment of the present invention discloses a real-time PCR- based amplification kit for quantitative detection of IHHNV in a biological sample, comprising a primer pair having nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2; a fluorescent probe having a nucleotide sequence set forth in SEQ ED NO: 3; and a PCR master mixture.
• the primer pair and the fluorescent probe are designed to specifically recognize and amplify a conserved ORFl in the IHHNV genome; whereas the fluorescent probe is preferably having a reporter fluorescent FAM dye at its 5' end and a non-fluorescent MGB quencher at its 3' end.
• the TaqMan-based real-time quantitative PCR assay is developed by incorporating the fluorescent TaqMan probe into the 5' nuclease PCR assay.
• the established IHHNV real-time assay can be evaluated for its sensitivity and specificity.
• the method and kit invented promises high-throughput screening program, accompanying with high sensitivity, specificity and reproducibility.
• this IHHNV real-time technique is also proven to be feasible in monitoring the viral infection in shrimps of different origins and at different development stages, even in their larval stages, for both infected shrimps or healthy pathogen-carrying shrimps.
• Figure 1 is the list of nucleotide sequences of the primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) and the probe (SEQ ID NO: 3) which are applied in the real-time PCR quantitative detection of the IHHNV as described by one of the preferred embodiments of the present invention.
• Figure 2(A) is a logarithmic amplification plot of the real-time IHHNV qPCR sensitivity test with a series of 10-fold dilutions of purified plasmid DNAs as described by one of the preferred embodiments of the present invention, showing the standard curve of the logarithm of IHHNV plasmid copy versus threshlod cycle value. The data points represent each plasmid standard prepared in triplicate. The correlation of coefficient, R 2 and the linear regression equation are shown.
• Figure 2(B) is the logarithmic plot of real-time PCR amplification of delta Rn
• Rn normalized reporter
• delta Rn Rn minus baseline.
• the curves represent the amplification event of plasmid standards in triplicate, wherein the copy numbers of the standards were denoted on the plot.
• Figure 3(A) is the logarithmic amplification plot of (a) 1 x 10 9 copies of IHHNV plasmids, (b) 10 copies of IHHNV plasmids, (C) NTC, (d) HPV- infected prawn sample, and (e) genomic clones of MrNV, as described by one of the preferred embodiments of the present invention.
• Figure 3(B) is a electrophoresed gel of real-time PCR products, in which lane M:
• the present invention relates to a method for detecting viral infection in shrimps which is caused by the infectious hypodermal and haematopoietic necrosis virus (IHHNV). More particularly, the present invention provides a high-throughput realtime viral detection method for screening and identifying IHHNV in shrimps, as well as a rapid detection system and kit thereof; which are capable of controlling the prevalence of the IHHNV in shrimps, thus enhancing the breeding program in the shrimp-farming industry, especially for giant fresh water prawn, Macrobrachium rosenbergii.
• the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.
• the present invention discloses a method for quantitatively detecting IHHNV in a biological sample, comprising the steps of obtaining a primer pair having nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, and a fluorescent probe having a nucleotide sequence set forth in SEQ ID NO: 3; amplifying a non-structural protein coding region of the virus in the biological sample by a real-time PCR using the primer pair and the fluorescent probe; and determining the presence and quantity of the virus in the biological sample with reference to a positive control plasmid.
• Figure 1 shows the list of nucleotide sequences of the primer pair and the fluorescent probe designed and constructed for use in the real-time PCR quantitative detection of the IHHNV as described by one of the preferred embodiments of the present invention.
• SEQ ID NO: 1 refers to the nucleotide sequence of the forward primer flanking the non- structural protein coding region of the IHHNV
• SEQ ID NO: 2 refers to the nucleotide sequence of the reverse primer.
• the fluorescent probe provided has a nucleotide sequence as set forth in SEQ ID NO: 3.
• the method for quantitatively detecting the presence and quantity of IHHNV in the biological sample can be initiated with a shrimp sample collection process.
• the biological sample is preferably derived from tissues or whole bodies of an aquatic animal collected at different development stages from different growth environments.
• the aquatic animal is a shrimp species of M. rosenbergii.
• Shrimp samples of this species can be collected at their different development stages, including adult, juvenile and larval stages, from different growth environments, including river, hatchery and growout.
• An example of the shrimp sample collection process is further detailed in Example 1.
• the shrimp sample collected can be an asymptomatic viral pathogen carrier or an individual infected with the disease.
• the biological sample is a DNA or cDNA template derived from the RNA of the sample.
• a simple technique for extracting the DNA is further detailed in Example 2, in which the commercially available lysis buffer can be applied to lyse the sample issues or whole bodies of the shrimp samples.
• cDNA can be obtained from a reverse-transcription procedure if RNA is extracted.
• a primer pair and probe designing process is preferably conducted before the real- time PCR-based amplification process.
• the primer pair is designed from a nonstructural protein coding region of the IHHNV genome.
• Example 3 provides a method for designing the primer pair and the probe sequences.
• the IHHNV genome can be obtained from publicly available database, such as GenBank.
• GenBank the primer and probe sequence designing process can then be facilitated by the publicly available software in the internet.
• the primer pair and the fluorescent probe are specifically designed to recognize and amplify a 65 bp DNA fragment originated from the conserved ORF1 of the non-structural protein coding region in the IHHNV genome.
• Still another preferred embodiment of the present invention discloses a technique for constructing the fluorescent probe for the real-time PCR detection.
• the fluorescent probe which is commercially known as TaqMan probe is preferably constructed.
• the probe is synthesized with a reporter fluorescent dye at its 5' end and a non-fluorescent quencher at its 3' end. More preferably, the reporter fluorescent dye is FAM; whereas the non-fluorescent quencher can be MGB.
• the present invention does not intend to limit the use of other types of fluorophore besides FAM, as long as they are suitable to be applied in the real-time PCR assay. Similarly, different types of non-fluorescent quencher can also be applied in the present invention.
• a positive control plasmid is also required in the real-time PCR detection of the present invention. It is disclosed in yet another preferred embodiment of the present invention that the positive control plasmid is obtained from a competent cell which is transformed with a vector cloned with a 389 bp region of the IHHNV genome.
• the 389 bp region of the IHHNV can be obtained from a PCR amplicon amplified using the forward and reverse primers (389F/R) listed in the ⁇ manual.
• Appropriate types of cloning vector can be employed as long as they are compatible with the competent host cell.
• An agarose gel electrophoresis can be conducted to confirm the plasmid which is isolated from the host cell, after the transformation and sub-culturing.
• the purified plasmid can be further sequenced to ensure the presence of the correct nucleotide sequence.
• concentration of the positive control plasmid constructed can also be determined for future application, including the quantitative detection of IHHNV virus at a later stage.
• the real-time PCR amplification process can be performed according to the protocol as further described in Example 5.
• the presence and quantity of the IHHNV in the biological samples tested can be determined from the sigmoid plot generated by the real-time PCR assay, with reference to the positive control plasmid.
• a primer pair for use in a real-time PCR-based quantitative detection of IHHNV in a biological sample, comprising nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2.
• the primer pair designed can be constructed commercially.
• Another further embodiment of the present invention is a fluorescent probe for use in a real-time PCR-based quantitative detection of IHHNV in a biological sample, comprising a nucleotide sequence set forth in SEQ ID NO: 3.
• the primer pair and the fluorescent probe are preferably designed to specifically recognize and amplify a conserved ORFl of a non-structural protein coding region in the IHHNV genome.
• the fluorescent probe is having a reporter fluorescent FAM dye at its 5' end and a non-fluorescent MGB quencher at its 3' end.
• Still another further embodiment of the present invention discloses a real-time PCR- based amplification kit for quantitative detection of IHHNV in a biological sample, comprising a primer pair and a fluorescent probe as set forth in the foregoing description; and provided with a PCR master mixture.
• the PCR master mixture can contains the typically used PCR enzyme and buffer solution.
• the PCR master mixture of the amplification kit of the present invention contains a pre- determined amount of magnesium chloride, deoxyribonucleotide triphosphate, Taq DNA polymerase and Taq buffer with potassium chloride.
• the fluorescent probe is preferably having a reporter fluorescent FAM dye at its 5' end and a non-fluorescent MGB quencher at its 3' end.
• sequence-specific TaqMan probe is especially appealing in the real- time detection.
• the fluorecence of the FAM dye in the intact TaqMan probe is absorbed by the MGB quencher in close proximity. This is then followed by cleavage of the probe by 5' ⁇ 3' exonuclease activity of Taq polymerase during primer-extension phase that spatially separates the FAM reporter dye and MGB quencher dye, resulting in fluorescent signal production.
• the release of FAM reporter fluorophore from the internal hybridizing probe and the resulting increase in fluorecence signal during amplification cycle is thus in proportion to the number of amplicons generated.
• the established IHHNV real-time PCR assay can be evaluated for its sensitivity and specificity. An example of the evaluation test is further detailed in Example 6.
• the sensitivity of real-time TaqMan qPCR can be determined using a series of 10-fold dilutions of the purified plasmid constructed. As revealed by the experimentation, the method and system provided by the present invention can have a detection limit of 10 DNA copies. A highly reproducible log-linear range up to 1 x 10 s copies can also be proven.
• the specificity of the real-time primer pair and the fluorescent probe can be tested on biological samples, preferably DNA samples, extracted from shrimp samples which are infected by other types of viruses.
• the primers and the probe are found to be specific to only IHHNV and do not cross react with genomic seqences of, such as hepatopancreatic parvovirus (HPV) and M. rosenbergii nodavirus (MrNV).
• the method and kit of present invention can be used in quantitative analysis of IHHNV in M. rosenbergii sampled at different development stages and from different locations.
• the method and kit invented have been proven to be able to detect up to 10 3 copies ⁇ g "1 DNA in wild berried female adults, larvae nursed at hatchery, and juveniles reared in farm for 6 weeks.
• the 10-fold to 100-fold increases in viral titre equivalent to 10 3 to 10 4 copies g "1 IHHNV DNA can be shown in sub-adult prawns reared in farm for 15 weeks.
• the real-time qPCR method and kit of the present invention promises rapidity and accuracy in quantitative detection of IHHNV.
• a wide range of template amounts can be quantified at high accuracy and reproducibility using the real-time IHHNV PCR assay.
• the primers and the probe are also specific to IHHNV DNA, without exhibiting cross-reactivity with the genome sequences of other viruses and shrimp hosts.
• the wild-caught broodstock from the same source were also spawned at the local prawn hatchery centre.
• the larvae produced from the wild female at the hatchery were later sampled for study.
• the remaining larvae were nursed through to post-larval stage, at which time point the shrimps were delivered to a private, locally-operated farm in Kuala Kangsar, Malaysia, for further development into juveniles and sub- adults.
• the juvenile and sub-adult prawn samples were collected at 6 and 15 weeks, respectively, following the transfer.
• the specific primers and probe sequences for quantitative detection of IHHNV were designed from a non-structural protein coding region (ORF1) of IHHNV genome in GenBank AF218266 using Primer Express software version 3.0 (Applied Biosystem, Foster City, CA, USA).
• the primers generate amplicon of 65 bp.
• the internal TaqMan probe was synthesized with a reporter fluorescent dye 5-carboxyfluorescein (FAM) attached to the 5' end and a non-fluorescent minor groove binder (MGB) quencher attached to the 3' end.
• FAM reporter fluorescent dye 5-carboxyfluorescein
• MGB non-fluorescent minor groove binder
• the 389 bp PCR amplicon amplified using the forward and reverse (389F/R) listed in the OIE manual was cloned in the yT&A vector and designated as pTA-IHHNV.
• the plasmid DNA was transferred into ECOSTM 101 competent cells (Yeastern Biotech) and cultured in selective Luria Bertani agar medium containing ampicilin and X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactopyranoside). A single isolated white colony on agar plate was selected and the plasmids were purified using the HighYield Plasmid Mini Kit (Yeastern Biotech).
• the plasmid preparation was further treated with plasmid-safeTM ATP-dependent DNase (Epicentre Biotechnologies, Madison, WI, USA) to selectively eliminate linear double-stranded DNA resulted from bacterial genomic DNA contamination.
• plasmid-safeTM ATP-dependent DNase Epicentre Biotechnologies, Madison, WI, USA
• an aliquot of plasmid DNA was subjected to electrophoresis on a 1% EtBr-agarose gel and photographed using AlphalmagerTM (Alpha Innotech).
• the purified plasmid was sequenced to ensure the presence of correct nucleotide sequence and the concentration was determined by measurement of optical density at 260 nm.
• Real-time qPCR was performed in a 25 ⁇ reaction volume comprising of 0.15 uM of each IHHNV primers and TaqMan probe, 10 ng DNA, and IX Taqman universal PCR master mix optimized with AmpliTag Gold DNA polymerase, dNTPs with dUTP, ROX Passive Reference dye, and optimized buffer components (Applied Biosystems).
• Real-time amplification was carried out with 7500 Real-Time PCR System (Applied Biosystems) using the following thermal cycling conditions: 10 min activation of AmpliTaq Gold at 95 °C, followed by 40 cycles of 15 s denaturation at 95 °C and 1 min annealing and extension at 60°C.
• the sensitivity of real-time TaqMan qPCR was determined using a series of 10-fold dilutions of the purified plasmid, pTA-IHHNV.
• concentrations of plasmid standards ranging from 10 copies to 10 9 copies were tested.
• the SDS software was used to generate the standard curve with average Ct values for triplicate standards.
• An IHHNV-specific standard curve was generated by 10-fold serial dilutions of the plasmid standards.
• the logarithmic amplification plot and the standard curve are shown in Figure 2, demonstrating the detection limit at 10 copies of IHHNV DNA and the log-linear range up to lxlO 9 copies.
• HPV DNA, genomic clones of MrNV, and no-template control (NTC) displayed nagative results (c-e, Figure 3A) and no band was observed in agarose gel electrophoresis (2-4, Figure 3B).
• the primers and TaqMan probe were proven to be specific to IHHNV.

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