FIELD OF INVENTION

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.

BACKGROUND OF THE INVENTION

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. However, 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. To a more severe extend, 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. rosenbergii, which has been listed as one of the most significant aquacultural diseases by the World Organization for Animal Health (ΟΓΕ). This IHHNV infection has also been shown in the freshwater prawn, M. rosenbergii in Malaysia, and reported in the year of 2009. The infection was initially detected using the standard ΟΓΕ-listed primers which amplifies specific region of 389bp within the viral genome through conventional polymerase chain reaction (PCR) (OIE, 2003). However, the use of this suggested primer along with the existing experimental procedure revealed nonspecific band in the PCR result. It may be caused by the mutations occurred in the IHHNV viral genome, or the low level of viral loads in the samples. Therefore, one major interest in the shrimp-farming of M. rosenbergii is centred on the research and development of a more specific primer and a more effective method to obtain a more accurate and reliable detection result for IHHNV.

There are a few patented technologies over the prior art relating to methods, systems or kits for detecting IHHNV. Amongst these existing technologies, there is a U.S. Patent No. 2008293037 disclosing primer sequences diagnostic for the detection of the IHHNV. According to this document, the primers isolated are based on a new portion of the IHHNV genome and may be used in primer-directed amplification or nucleic acid hybridization assay method. This method is meant for detecting the presence of IHHNV and for quantifying the amount of this virus. However, this patented technology merely uses simple amplification and hybridization method but does not disclose any specific PCR primer together with its predetermined procedure which is able to provide a rapid and specific detection result. 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. However, this nested PCR method may not be sufficiently sensitive to detect the presence of IHHNV in shrimps at its development stage.

Apart from that, 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. However, this document does not disclose the origin of the IHHNV primers applied. There is also no disclosure provided by this document on the efficiency of this method and kit, whether the IHHNV of different species can be quantitatively detected at their different development stages.

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. Besides, quantification of viral load in infected prawns also allows the monitoring of cultured shrimp disease and hence effective measures can be taken before the disease outbreak. As revealed by the prior art, the currently existing PCR-based detection of IHHNV are not capable of giving a specific and reliable result, which can be used to sensitively and accurately detect IHHNV in M. rosenbergii, especially at its various development stages. In addition, most of the detection methods and systems available in the prior art are focused on the infected shrimps and not the healthy carriers. Hence, it is desirable for the present invention to provide a specifically-designed method, system or kit for detecting IHHNV quantitatively in real-time to overcome the drawbacks of the prior art.

SUMMARY OF INVENTION

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.

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.

At least 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.

According to a preferred embodiment of the present invention, 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. In accordance with another preferred embodiment of the present invention, the aquatic animal is a shrimp species of M. resenbergii; and the different development stages preferably include adult, juvenile and larval stages. Still another preferred embodiment of the present invention discloses that the primer pair and the fluorescent probe are designed from a non-structural protein coding region of the IHHNV genome.

Yet another preferred embodiment of the present invention discloses that the fluorescent probe is synthesized with a reporter fluorescent dye at its 5' end and a non- fluorescent quencher at its 3' end. Preferably, the reporter fluorescent dye is 5- carboxyfluorescein (RAM); whereas the non-fluorescent quencher is minor groove binder (MGB). 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.

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.

Preferably, 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. As set forth in the preceding embodiments, 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. Preferably, 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.

In order to enhance the rapidity, sensitivity and specificity of the viral detection system, 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. Besides, 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. With the accurate information provided by the real-time assay, adequate prevention measures can thus be taken to avoid outbreaks of viral disease. Further, with the high sensitivity of the method and system invented, the development of specific pathogen free (SPF) shrimps can be achieved and this can fulfill the current demand in various breeding programs for producing and maintaining high quality domesticated viral-free broodstocks for sustainability of food source and environment biosafety, in accordance with the food safety and environment regulations in the world.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

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

versus the PCR cycle numbers, in which the baseline cycle range was set between cycle 3 to 11. Rn (normalized reporter) is the ratio of fluorescence intensity of reporter dye to fluorescence intensity of the ROX passive reference dye; delta Rn is 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:

20bp DNA ladder marker; lane 1: 1 x 10 ⁹ copies of plasmid DNAs; lane 2: NTC; Lane 3: HPV DNA samples; land ane 4: genomic clones of MrNV.

DETAILED DESCRIPTION OF THE INVENTION 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. Hereinafter, 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. Accordingly, SEQ ID NO: 1 refers to the nucleotide sequence of the forward primer flanking the non- structural protein coding region of the IHHNV whereas SEQ ID NO: 2 refers to the nucleotide sequence of the reverse primer. Whilst, the fluorescent probe provided has a nucleotide sequence as set forth in SEQ ID NO: 3.

In accordance with the preferred embodiment of the present invention, 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. In accordance with the preferred embodiment of the present invention, 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.

According to a preferred embodiment of the present invention, 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. Optionally, 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. In accordance with still another preferred embodiment of the present invention, 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. Accordingly, the IHHNV genome can be obtained from publicly available database, such as GenBank. The primer and probe sequence designing process can then be facilitated by the publicly available software in the internet. Preferably, 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. Preferably, the fluorescent probe which is commercially known as TaqMan probe is preferably constructed. According to the preferred embodiment, 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. However, 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.

Apart from the primer pair and the fluorescent probe, 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. As an example, as further described in Example 4, 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 ΟΓΕ 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. Besides, the 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.

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. 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. As set forth in the preceding description, 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. More preferably, 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. Preferably, 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.

As set forth in the preceding description, 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 use of sequence-specific TaqMan probe is especially appealing in the real- time detection. In the present invention, 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. Whilst, 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 μg ^"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. Besides having a high level of sensitivity, the wide dynamic log-linear quantification range of 9 orders of magnitude established in this real-time PCR assay with high correlation of coefficient (R ² >0.99). 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 present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

EXAMPLE

Examples are provided below to illustrate different aspects and embodiments of the present invention. These examples are not intended in any way to limit the disclosed invention, which is limited only by the claims.

Example 1 Prawn samples collection

M. resenbergii prawn samples collected at diferrent development stages (adult, larva and juvenile) and growth environments (river, hatchery and growout) in Perak, Malaysia, were used in the quantitative study. Three asymptomatic individuals of wild berried female broodstock were initially obtained from Rubana River for IHHNV analysis. 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.

Example 2 DNA extraction

Several pairs of pleopods from juvenile, sub-adult and adult prawns were cut and fixed in absolute ethanol prior to DNA extraction. For assay of larvae, whole bodies were used. Fifteen to twenty larvae of the same populations were pooled into a single tube. The sample tissues or whole bodies were lysed with the lysis buffer from IQ2000™ diagnostic kit (Farming IntelliGene Technology Corporation) according to the manufacture's instruction. Cell lysis was followed by isopropanol precipitation and subsequently DNA pellet dissolution in 50 μΐ TE buffer. Example 3 Real-time PCR primers and probe design

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. The sequences of the primers and probe and their relative nucleotide position in the genome AF218266 are shown in Table 1.

Table 1

Primer/ Probe Sequence (5' - 3') Nucleotide

Position (AF218266)

Forward primer GAC ACC CAA CCA ATA AGA CCA GA 1615-1637

Reverse Primer GC A GCA AAG GTA ACT CCC AAA T 1660-1681

Probe ATA GAG CTA CAA TCC TCG CC 1639-1658 Example 4 Construction of positive control plasmid

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 ECOS™ 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-safe™ ATP-dependent DNase (Epicentre Biotechnologies, Madison, WI, USA) to selectively eliminate linear double-stranded DNA resulted from bacterial genomic DNA contamination. To confirm purity, an aliquot of plasmid DNA was subjected to electrophoresis on a 1% EtBr-agarose gel and photographed using Alphalmager™ (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.

Example 5 Real-time quantitative PCR assay

Real-time qPCR was performed in a 25 μΐ 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. Real-time fluorescent measurements were monitored with a built-in charge-coupled device (CCD) and data analysis was performed using the SDS software version 1.3.1. The threshold cycle (Ct) value was automatically defined by the software as standard deviations of baseline emission for cycles 3-15. The baseline cycle range was changed accordingly when Ct value of most abundant sample or standard was lower than 15 cycles. The real-time PCR assay has a cut-off value of 40 cycles. Samples that were characterized with sigmoid plot and Ct < 40 were interpreted as positive, and vice versa. To confirm the assay results, 4% EtBr-agarose gel electrophoresis was performed. Example 6 Sensitivity and specificity of real-time PCR

The sensitivity of real-time TaqMan qPCR was determined using a series of 10-fold dilutions of the purified plasmid, pTA-IHHNV. The 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. The real-time TaqMan assay has a dynamic quantification range of 9 orders of magnitude and the standard curve over this range has coefficient of regression more than 0.99 (R ² = 0.998) with the standard deviation of the triplicate plasmid standard at each concentration no more than 0.23, as shown in Table 2. the results demonstrated that the linear, 9-log, relationship between the Ct value and the number of copies of IHHNV is reproducible and statistically significant.

Table 2

Copy number of IHHNV plasmid Average Ct Standard deviation

10 37.37 0.23

50 35.69 0.02 lxlO ² 34.59 0.10 lxlO ³ 31.59 0.12 lxlO ⁴ 28.26 0.11 lxlO ⁵ 24.16 0.18 lxlO ⁶ 20.65 0.05 lxlO ⁷ 17.09 0.03 lxlO ⁸ 13.46 0.04 lxlO ⁹ 11.03 0.05 The specificity of the real-time primers and probe was tested on DNA samples extracted from HPV-infected M. rosenbergii and on plasmid clones (pET) containing MrNV whole genome (Lab-Ind Resource & UPM, Malaysia). Amplification event was occurred only in IHHNV plasmid DNA (a and b, Figure 3A; 1, Figure 3B). 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.

Three samples from each development stage were quantified by the real-time TagMan PCR assay. The results showed that all samples collected and cultured at different locations were infected with IHHNV, as shown in Table 3. The DNA extracted from the pleopods of wild-caught berried female and from the whole bodies of larvae contained quantities of IHHNV DNA ranging from 2.9 x 10 ³ to 5.2 x 10 ³ copies μg ^"1 DNA and 2.1 x 10 ³ to 3.4 x 10 ³ copies μ^ ¹ DNA, respectively. The IHHNV quantities in the 6-week farm-reared juvenile and 15-week farm-reared sub-adult prawns were up to 9.5 x 10 ³ copies DNA and 5.0 x 10 ⁴ copies μg ^"1 , respectively.

Table 3

Copy no. ug ^"1 Type of tissues Origin Development Stage Sample DNA

2.9 x 10 ³ Pleopods Wild-caught Berried adult female 1

5.2 x 10 ³ Pleopods Wild-caught Berried adult female 2

3.0 x 10 ³ Pleopods Wild-caught Berried adult female 3

2.1 x 10 ³ 15-20 whole bodies Hatchery Larva 4

3.4 x 10 ³ 15-20 whole bodies Hatchery Larva 5

2.8 x 10 ³ 15-20 whole bodies Hatchery Larva 6

2.2 x lO ³ Pleopods Pond Juvenile 7

2.4 x 10 ³ Pleopods Pond Juvenile 8

9.5 x 10 ³ Pleopods Pond Juvenile 9

5.0 x 10 ⁴ Pleopods Pond Sub-adult 10

1.2 x 10 ^s Pleopods Pond Sub-adult 11

3.8 x 10 ⁴ Pleopods Pond Sub-adult 12