Ghrissi, M.

Mohd Lila, Mohd Azmi

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1998: Volume 2DEVELOPMENT OF NUCLEIC ACID PROBES FORTHE DIAGNOSIS OF CHICKEN ANEMIA INPOULTRYM.L. Mohd-Azmi and M. GhrissiFaculty of Veterinary MedicineUniversiti PutTa Malaysia, 43400 UPM, Serdang, Selangor,MalaysiaKeywords: chicken anaemia virus, DNA probe,ELISA,PCR.IntroductionChicken anaemia virus (CA V) is a single positive strandedDNA virus, which transiently causes severe anaemia andimmunodeficiency in young chicks and subclinical in adults).Conventionally, diagnosis of CAV infection can be con-ducted by virus isolation of the detection of CAV-specificantibodies. However, these methods are cumbersome, lesssensitive and time consuming that impairs the rapid and reli-able laboratory diagnosis with clinical samples. An alterna-tive diagnostic approach, polymerase chain reaction (PCR)technique, is known to be an extremely sensitive assay.Combination of PCR technique with ELISA technique willfurther improve the reliability of the diagnostic method. Thisresearch was designed DNA probes together with a reliablediagnostic process for the diagnosis and screening of chick-ens infected by CAV .Materials and MethodsChickens infected by CAV were identified by the use ofELISA. Infected chickens were sacrificed and thymus andliver were collected. These tissues were used as source ofCAV DNA. Three pairs of oligonucleotide primers were de-signed and synthesised. These primers were used in PCR tospecifically amplify CAY genes: VPI, VP2 and VP3 genes.These genes were cloned individually into plasmid vectorsand subjected for DNA sequencing. The homology of thesegenes with various CAV strains were determined by se-quence alignment computer software. DNA sequences thatmostly unique to individual genes and the virus were deter-mined. The DNA sequences selected were used to design asingle unit of DNA probe namely dVPl, dVP2 and dVP3.The design could be expanded in combination with two orthree units of probes of individual VPl, VP2 and VP3 genes.A single unit of individual probes was synthesised. A biggersingle unit of the probe containing at least 50 tandem-repeatswas constructed by the standard cloning method by usingplasmids. Such recombinant plasmid could be used as aprobe for detection of multiple short DNA targets by meansof ELISA. Single stranded DNA probe could be prepared ina phagemid vector. The DNA target must first be obtained bythe use of any PCR primers that can be used to amplify CAVDNA spanning the VP1, VP2 or VP3 gene.Results and DiscussionThree sets of primers have been designed and found to bespecific for chicken anaemia virus (Ghrissi and Mohd-Azmi,1998). The entire VPl, VP2 and VP3 genes of CAY havebeen cloned into bacteria plasmid vectors (Raha et al. 1997).The VP3 gene has been fully sequenced and deposited intoGENEBANK. The size of the VP3 gene was determined tob104372 bp and its DNA sequence could be accessed with acces-sion number AF030518 (Mohd-Azmi et al. 1997). The VP3gene has been further cloned in eukaryotic expression vector.In related studies, the VP3 gene has been expressed in bacte-ria and mammalian cell lines. It showed that the VP3 genehas anticancer properties which were potentially not limitedto avian cells only. The VP2 and VP3 genes cloned have alsobeen sequenced. The DNA sequences of the three genes havebeen analysed for the preparation of DNA probes. Single unitDNA probes were prepared for the individual genes. Resultsshowed that the single unit probe dVPl against VPl genewas highly specific compared to the other two probes. How-ever, combination of two probes, dVPI with either dVP2 ordV:P3, promotes both specificity and sensitivity. The singleumt probe dVP1 was used to prepare tandem array of single-stranded DNA probe. Tandernly repeated DNA units of dif-ferent length were successfully prepared by ligation methodwith DNA ligase and cloning. Further treatment and self-ligation to identical units increased the overall length of theprobe. Various sizes of probes were cloned into plasmidpSPORT. Bacteria Escherichia Coli JMI09 were trans-formed with the recombinant plasmid and co-infected withphage M13K07. Phage particle containing ssDNA probewere purified and used as an immobilised probe in ELISAplate. The probe was then hybridised to the solid phase bymeans of uv-cross-linking, The immobilised probes were re-acted with PCR products that contained the specific portionVPl gene. Primers used in the PCR were labeled with biotin.Results of such hybridisation showed that the maximum sig-nal as detected in ELISA could be reached when the numberof tandem repeats per unit probe was reaching approximately50 copies. The signal was not significantly improved whenthe number of repeats further increased. Furthermore, whenthe length of probe was increased further, the instability re-combinant plasmid and decreased in recovery of ssDNAwere observed. The detection limit is defined as the mini-mum amount of PCR amplified product of target samplesrequired for the detection system to work and its is veryminimum i.e., 1ng. The technique developed here is simpleand highly sensitive to detect PCR-amplified of CAV DNAsamples.ConclusionsThe DNA probe developed is very sensitive and useful forthe development of a full working diagnostic kit for chickenanaemia virus infection. The use of an appropriate DNAbinding protein to bind the DNA probe to the ELISA platewould be of value for the preparation of low-cost diagnostickit.ReferencesGhrissi, M. and Mohd-Azmi, M.L. 1998. Polymerase chain reaction(PCR) Amplification and Molecular Cloning of VP2 Gene ofMalaysian Chicken Anemia Virus (CAV). Proc. 10th NationalBiotechnology Seminar, 27-28 Oct. 1998, Shah Alam. p. 283-285.Mohd-Azmi, M.L., Ghrissi, M., Raha, R.A., Yusof, K. and Aini, I.1997. A complete DNA sequence of the VP3 Gene of ChickenAnaemia Virus (Malaysian Isolate). Gene Bank. Accession Num-ber AF030518.Raha, .A. R, Yusof, K, Sipat, A.B., Omar, A.R., Mohd-Azmi, M.L.,Ahmed, N. and Ng, P.T. 1997. Cloning and sequence of majorVP genes of chicken anaemia virus genome. Proc. 9th NationalBiotechnology Seminar, 22-26 Nov. 1997, Penang. p. 231-233.Supported by [RPA Grant 0[-02-04-0093

Development of nucleic acid probes for the diagnosis of chicken anemia in poultry

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Development of nucleic acid probes for the diagnosis of chicken anemia in poultry

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