Development Of Sybr Green I Real-Time Pcr Method For Detection And Differentiation Of Newcastle Disease Virus Pathotypes

Newcastle disease (ND) which is caused by Newcastle disease virus (NDV) is a highly contagious viral disease of domestic poultry, cage, aviary and wild birds. ND outbreaks have led to substantial losses in the poultry industry. NDV can be classified into three major pathotypes: velogenic, mesogenic and lentogenic. Velogenic strains are highly virulent and may lead to 100% mortality in infected chicken whilst mesogenic and lentogenic strains cause mild clinical or inapparent infections, respectively. Early detection and differentiation of NDV pathotypes are very important during monitoring of suspected ND cases or during disease outbreaks. In this study, SYBR Green I real- time polymerase chain reaction (PCR) was developed for detection and differentiation of NDV pathotypes. Velogenic-specific primers (NDVIF2 & NPV2N) and lentogenic-specific primers (NDVIF2 & NPL2N) were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. After establishing the optimum condition of the real-time PCR, the assay was performed on 22 previously characterized NDV strains. All the velogenic strains were only detected by using velogenic-specific primers (NDVIF2 & NPV2N) with threshold cycle (Ct) ranged from 12.92 to 22.76 and melting temperature between 85.6°C to 86.4°C. Similarly, all the lentogenic/vaccine strains were only successfully detected when lentogenic-specific primers (NDVIF2 & NPL2N) were used. All the lentogenic/vaccine strains amplified with the lentogenic-specific primer had a Ct value ranged from 11.93 to 18.73 and Tm between 87.2°C to 87.6°C. No amplification was found when the NDV velogenic-specific primers and lentogenic-specific primers were used to amplify avian influenza virus (AIV), infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV). This revealed that both velogenic- and lentogenic-specific primers were pathotype specific and no unrelated viral RNA can be amplified. The newly developed assay had a dynamic detection limit which spans over a 5 log10 concentration range. The velogenic and lentogenic amplifications showed high PCR efficiency of 98.8% and 103%, respectively. Mean coefficient variation (CV) of reproducibility tests for velogenic amplification and lentogenic amplification was around 1% and 2%, respectively. The SYBR Green I real-time PCR was 10-fold more sensitive when compared to the conventional detection method using agarose gel electrophoresis. Turnaround time for the developed assay was approximately 2.5 hours including reverse transcription, PCR amplification and melting curve analysis. Clinical samples from the experimental infected chickens as well as the suspected field cases were collected and then tested on the developed assay. In the experimental infection with lentogenic NDV F strain, virus could be detected 3 days post infection (p.i.), followed by day 4, 5 and 10 p.i. For the SPF chickens infected with high doses of velogenic NDV strain AF2240 (105 to 103 ELD50/0.1 ml), the virus can be detected as early as day 2 p.i., followed by day 3 and 4 p.i. All the infected chickens were dead on day 4 p.i. For the chickens group infected with low doses (102 to 100.5 ELD50/0.1 ml), the virus can be detected starting on day 4 p.i., followed by day 5, 7, 10, 11 and 12 p.i. All the infected chickens were dead on day 12 p.i. The assay was able to detect the viruses as early as day 2 before the observation of clinical signs. This is an important achievement as early detection can prevent further spread of the disease. A total of 41 suspected NDV field cases were tested with the developed assay, 33 cases were NDV negative and 8 cases were positive for velogenic NDV. The results were correlated well with the virus isolation method and F cleavage site sequence analysis. All these 8 isolates possess two pairs of dibasic amino acids at the position 112 to 116 of the F cleavage site, and a phenylalanine residue at the position 117. This F cleavage site analysis revealed that all of the 8 NDV isolates belonged to velogenic group. In the attempt to improve the efficacy of the developed assay, internal amplification control (IAC) was incorporated into the developed real-time PCR assay for detection of PCR inhibitors. The potential of simultaneous detection of IAC and NDV target was investigated. The simultaneous detection was achieved based on the melting curve analysis. The co-amplified products exhibited two distinguished melting peaks at 86.36±0.13°C and 91.42±0.21°C which corresponded to NDV NP gene product and IAC KanR gene product, respectively.In conclusion, this study successfully developed a SYBR Green I real-time PCR for NDV pathotypes detection and differentiation. The virus can be detected directly from clinical samples without the need of virus propagation in chicken embryonated eggs. Owing to these advantages, the developed assay will contribute significantly in the control and prevention of the spread of the disease. ND-infected birds can be rapidly isolated from the healthy bird in the case of field outbreaks, if the causal agent is detected at the early stage of the outbreak. Consequently, spread of the disease and economical losses can be prevente

Characterisation of Newcastle Disease Virus (NDV) Isolates and Development of Real-Time PCR for Diagnosis of NDV

A sudden upsurge of Newcastle disease (ND) outbreaks occurred in Malaysia with high mortality among vaccinated birds started in August 2000 and peaked between November 2000 and March 2001. Four isolates; 00/IKS, 0l/C, 01/TM and Ol/GNS were isolated from the NDV outbreaks in different states in Peninsular Malaysia. Mean death time (MDT) assay was carried out to determine the pathogenicity of the isolates in embryonated chicken eggs. All isolates had MDT of less than 60 hours, indicating that the isolates are velogenic. The nucleotide sequence in the region of F cleavage site for the four NDV isolates were determined and the deduced amino acid sequence showed that all isolates possessed two pairs of basic amino acids (112RRQKR116) and Phe residue at position 117 of the F cleavage site. These amino acid sequences correlated with the MDT results which confirmed that the four NOV isolates are velogenic strains. Analysis of the partial sequence of the F gene was carried out and the results suggested that the four recent isolates can be grouped under the genotype VII viruses. All the isolates possess K101 and V121 at the F gene sequence, a characteristic of genotype VII viruses. Isolates 0l/C had an N residue at position 101, which is unique among these four isolates. The phylogenetic analysis of the four isolates based on the partial sequence of M gene showed that their nucleotide similarities varied between 92.7% and 100%, where isolate 00/IKS shared 100% nucleotide sequence similarity with isolate Ol/GNS. The four isolates were found to be phylogenetic ally related to pigeon isolate 1307/US/75 and goose isolate ZJI with similarity ranging from 93.29% to 98.38%. This suggests that the recent isolates might have originated from domestic and free-living birds. There was no evidence to show that the recent isolates evolved from local velogenic NDY strain AF2240, which was isolated in 1960s. The nucleotide similarities of the four NDY isolates with strain AF2240 were 79.88% to 82.63%. Recent development of real-time PCR has offered the opportunity of developing a sensitive and accurate method to detect NDY. A two-step real-time RT-PCR procedure using the SYBR Green I dye was used as a detection signal. Beside the four isolates, 8 other isolates of NDV were used in this study. They were grouped into 7 velogenic, 1 meso genic and 4 lentogenic strains. All isolates showed positive results in amplification. A melting curve was obtained immediately after amplification to distinguish specific product from non-specific and primer-dimer. From the melting curve analysis, no primer-dimer and non-specific products were detected and all the isolates had melting temperature (Tm) ranging from 86°C to 87°C. The detection limits of the real-time PCR were compared with R T -nested PCR ELISA and agarose gel electrophoresis by preparing serially ten-fold dilutions of the cDNA. The real-time PCR could detect up to 1:10^5 dilution of cDNA with the concentration of 1.1 x 10-5 ug/ul. However, ELISA assay detection limit reached 1:103 with the concentration of 1.1 x10-3 ug/ul, whereas the agarose gel electrophoresis can only detect up to 1:10^2 dilution of the cDNA (1.1 x 10-2 ug/ul) with a faint band. The SYBR Green I real-time peR was found to be 100-fold more sensitive thari the peR-ELISA detection method. The study has therefore successfully developed a sensitive, rapid and convenient method for NDV diagnosis using real-time peR with SYBR Green I as a detection signal

Sequence analysis of the fusion(F) and matrix(M) genes of recen1ly isolated New Castle disease virus from Malaysia

The partial amino acid sequences of fusion (F) and matrix (M) genes encompassing the F cleavage site and the nuclear localization signals, respectively, of 4 recent Newcastle disease virus (NDV) isolates obtained from the field outbreaks between 2000 and 2001 were characterised. Isolates Ol/C, 01/TM and 01/GNS shared the same amino acid sequences at the F cleavage site 111GRRQKRF117.However, isolate OO/IKS has l1lERRQKRFI17 motif at the F cleavage site. Isolate 01/TM also has the highest number of amino acid substitutions at the F gene. Both isolates OO/IKS and Ol/GNS shared 100% amino acid sequence identity at the M gene whilst isolates Ol/C and 01/TM have 3 and 1 unique amino acid substitutions. All the 4 isolates also have 2 unique amino acid substitutions (R200RS and E213D). However, no particularly distinguishing sequence variations were identified among the different NDV pathotypes. All the 4 NDV isolates were grouped into genotype vn with bootstrap value of 90%. The genotype vn comprises the recent NDV strains recovered during the outbreaks between years 1995 and 2001 which have been reported in many countries including China, Taiwan, South Africa and Europe. As the 4 NOV isolates were found phylogenetically related to goose isolate ZJI and pigeon isolate 1307/US/75, they were placed in the same group with bootstrap value of 78 % based on the M gene sequence analysis

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10 ng of HBcAg with 108 pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples

The effects of conjugated linoleic acid isomers on the morphological changes in adipose tissue and adipogenic genes expressions on primary adipose tissue

Previous studies carried out in mouse 3T3-L1 cell culture have shown that conjugated linoleic acid (CLA) inhibited adipocyte differentiation. The present study was undertaken to investigate the effect of cis-9, trans-11 and trans-10, cis-12 CLA isomers on morphological changes and on mRNA expressions in the in vitro adipocyte isolated from specific pathogen-free (SPF) chicken. The adipocytes were isolated from SPF chicken and cultured in differentiation-induction medium with different concentrations of cis-9, trans-11 and trans-10, cis-12 CLA. After day 7, the adipocyte differentiations were monitored morphologically and mRNA expressions of lipoprotein lipase (LPL) and acyl-coenzyme A binding domain containing 5 (ACBD 5) were quantified by real-time polymerase chain reaction (PCR) analysis. Our data suggested that cis-9, trans-11 CLA downregulated the expression of LPL and ACBD 5 genes, which was concurrently observed with decrease in the adipocyte area size and cell number compared to the control and trans-10, cis-12 treated groups. Based on this finding, we concluded that dietary CLA modulate fat reduction in chicken via alteration of transcription of key adipogenic genes and adipose cellularity

Molecular detection and chracterization of infectious bronchitis virus from Libya.

Infectious bronchitis virus (IBV) is a very dynamic and evolving virus, causing major economic losses to the global poultry industry. Recently, the Libyan poultry industry faced severe outbreak of respiratory distress associated with high mortality and dramatic drop in egg production. Tracheal and cloacal swabs were analyzed for several poultry viruses. IBV was detected using SYBR Green I real-time PCR detection based on the nucleocapsid (N) gene. Sequence analysis of the partial N gene indicated high similarity (~ 94%) to IBV strain 3382/06 that was isolated from Taiwan. Even though the IBV strain 3382/06 is more similar to that of the Mass type H120, the isolate has been implicated associated with intertypic recombinant of 3 putative parental IBV strains namely H120, Taiwan strain 1171/92 and China strain CK/CH/LDL/97I. Complete sequencing and antigenicity studies of the Libya IBV strains are currently underway to determine the evolution of the virus and its importance in vaccine induced immunity. In this paper we documented for the first time the presence of possibly variant IBV strain from Libya which required dramatic change in vaccination program

Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (Ct) 18.19 ± 3.63 and a melting temperature (Tm) 86.0 ± 0.28 °C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the Ct value 14.70 ± 2.32 and Tm 87.4 ± 0.21 °C. The assay had a dynamic detection range which spans over a 5 log10 concentration range, 109–105 copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45 ± 0.31% and 1.30 ± 0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV

Preparation, optimization and swelling study of carboxymethyl sago starch (CMSS) - acid hydrogel

In this study, sago starch was modified in order to enhance its physicochemical properties. Carboxymethylation was used to introduce a carboxymethyl group into a starch compound. The carboxymethyl sago starch (CMSS) was used to prepare smart hydrogel by adding acetic acid into the CMSS powder as the crosslinking agent. The degree of substitution of the CMSS obtained was 0.6410. The optimization was based on the gel content and degree of swelling of the hydrogel. In this research, four parameters were studied in order to optimize the formation of CMSS–acid hydrogel. The parameters were; CMSS concentration, acetic acid concentration, reaction time and reaction temperature. From the data analyzed, 76.69% of optimum gel content was obtained with 33.77 g/g of degree of swelling. Other than that, the swelling properties of CMSS–acid hydrogel in different media such as salt solution, different pH of phosphate buffer saline solution as well as acidic and alkaline solution were also investigated. The results showed that the CMSS–acid hydrogel swelled in both alkaline and salt solution, while in acidic or low pH solution, it tended to shrink and deswell. The production of the hydrogel as a smart material offers a lot of auspicious benefits in the future especially related to swelling behaviour and properties of the hydrogel in different types of media

Molecular identification of species and production origins of edible bird's nest using FINS and SYBR green I based real-time PCR

The increasing demand and consumption of edible bird's nest (EBN) by people worldwide has contributed to the food fraud issue. To ensure the authenticity of EBN in regard to their origin, rapid and accurate analytical methods are very much needed. In this study, forensically informative nucleotide sequencing (FINS) technique based on mitochondrial and nuclear DNA sequences, and phylogenetic analysis was performed to identify the species and production origins of raw and commercial EBNs. The cytochrome b (Cyt b), NADH dehydrogenase subunit 2 (ND2), 12S ribosomal RNA and beta-fibrinogen intron 7 gene markers used were able to identify and classify EBN produced by Aerodramus fuciphagus and Aerodramus maximus. It was newly discovered that EBN from man-made houses and natural caves were genetically differentiable using the mitochondrial Cyt b and ND2 genes. The phylogenetic results revealed that all EBN samples were well-separated into two groups following their species origin and production origin. A rapid and cost-effective identification alternative of SYBR green I based real-time PCR assay targeting a 177 bp of the mitochondrial Cyt b gene was developed and it efficiently differentiated genuine EBN from counterfeits. This FINS and SYBR green I based real-time PCR are highly sensitive, specific and reliable methods for identification of EBN origins and could be useful for preventing fraud substitution and mislabelling of EBN to ensure food safety

Classification of entomological origin of honey based on its physicochemical and antioxidant properties

Physicochemical and antioxidant properties of raw honeys from Malaysia were used as markers for determining its entomological source of bee species of Apis dorsata, Apis mellifera, Apis cerana, or Heterotrigona itama. Physicochemical properties of moisture content, water activity, specific gravity, viscosity, pH, free acidity, electrical conductivity, colour (L*, a* and b*), colour intensity, and antioxidant properties including the DPPH free radical scavenging activity power (1/IC50), ascorbic acid equivalent antioxidant content (AEAC), ferric ion reducing antioxidant power (FRAP), and total phenolic content (TPC) were measured and analysed. Honeys were classified into two major groups of those from honey bees (Apis spp.) and Trigona stingless bees (Heterotrigona itama) from its physicochemical and antioxidant properties using hierarchical cluster and principal component analyses. The Kelulut honey produced by stingless bees, Heterotrigona itama was differentiable from honeys from the regular honey bee species, the Apis spp. with characteristics of high moisture content of 33.24 g/100 g, free acidity of 136.8 meq/kg, colour intensity of 990.3 mAU, AEAC of 26.64 mg/100 g, and FRAP of 41.95 mg AAE/100 g. Honey classification by its entomological origin helps in honey identification and it reduces honey fraudulence