[Diagnostic value of PCR method for detection of Salmonella enteritidis contamination in poultry products in Karaj]

Salmonella enterica serovar Enteritidis which involves poultry is transmitted by food. The aim of this survey was to optimize PCR method in order to detect Salmonella enteritidis in poultry products. In this basic research, 80 samples [40 broiler meat and 40 egg samples] were obtained from distribution centers in Karaj and suburb. Following the DNA extraction of the samples, PCR was optimized and performed using standard strain of Salmonella enteritidis [RTCC1621] as positive control and primers against the flagella coding sequence of Salmonella Enteritidis genome. The analysis of the PCR products by agarose gel electrophoresis indicated the amplification of 250 bp segments in 16 out of 40 [40%] broiler meat and 9 out of 40 [23%] egg samples. The sensitivity of the PCR at the DNA level was found to be 1pg and the specificity of the PCR was determined using 6 other entric Gram negative bacteria and found to be positive only for Salmonella enteritidis. This study confirmed that PCR provides sensitive, specific and rapid approach for detection of Salmonella enteritidis in food samples

Hepatitis B virus surface antigen variants clustered within immune epitopes in chronic hepatitis B carriers from hormozgan province, south of Iran

The aim of this study was to characterize the hepatitis B virus surface protein genotypes and sequence variations among hepatitis B virus surface antigen [HBsAg] positive chronic patients in Hormozgan province, south of Iran. A total of 8 patients enrolled in this study. The surface gene was amplified and directly sequenced. Genotypes and nucleotide/ amino acid substitutions were identified compared to the sequences obtained from the database. All strains belonged to genotype D. Overall 77 "mutations" occurred at 45 nucleotide positions, of them, 44 [57.14%] were silent [no amino acid altering] and 33 [42.86%] were missense [amino acid changing]. A number of 24 [80%] out of 30 amino acid changes occurred in different epitopes within surface protein, of which, 9 [30%] in B cell epitopes in 7 residues [2 occurred in "a" determinant region]; 8 [42.1%] in T helper epitopes in 7 residues and 7 [10%] in 4 residues inside CTL epitopes. Hepatitis B virus genome containing mutated immune epitopes no longer could be recognized by specific T-cells of the host immune surveillance and did not enhance anti-HBs production. This could led to the progression of chronicity B virus infection

Detection of bovine viral diarrhea virus in bovine semen using nested-PCR

A rapid and sensitive reverse transcription polymerase chain reaction [RT-PCR] and nested-PCR were used to detect bovine viral diarrhea virus 1 [BVDV-1] in bull semen. Selected primers could amplify a part of the 5'UTR of the BVDV genome. A 294 bp DNA fragment was amplified and specificity of the results was confirmed by direct sequencing of the PCR product. Prior to RNA extraction, the seminal inhibitors were eliminated using a simple dilution method. Therefore, a sensitivity of 3x102 50% tissue culture infective dose [TCID50] was achieved when experimentally infected semen was used for RNA extraction. In nested-PCR a 160 bp fragment was amplified and sensitivity of the test was increased to 3 TCID50. This technique can be used as a rapid and sensitive method of BVDV-1 detection in bovine semen

Differentiation of virulent and non-virulent newcastle disease virus isolates using RT-PCR

Newcastle disease is one of the main concerns of poultry farmers. Detection of virulent strains of Newcastle disease virus [NDV] has a great impact on control measures against the disease. In this study RT-PCR was optimized in high sensitivity in order to differentiate the virulent from non-virulent NDV isolates directly in tissue homogenates. The vaccinal NDV strain and known field isolates were tested by this technique. RT-PCR was performed using two sets of primers chosen from a section of the F gene. The PCR product was cloned in to a pTZ57R/T vector and sequenced. The sequence data confirmed the specificity of the test. Detection of viral virulence was determined based on the amplification of PCR products. The above optimized RT-PCR produce can be used to confirm the diagnosis of Newcastle disease within 24 hrs using RNA isolated directly from tissue homogenate or passaged in SPF [Specific Pathogen Free] embryonated eggs

Polymorphism of bovine lymphocyte antigen DRB3.2 in holstein bulls of Iran using PCR-RFLP

The Holstein bulls [n=50] were genotyped for bovine lymphocyte antigen [BoLA-DRB3.2] alleles by polymerase chain reaction and restriction fragment length polymorphism [PCRRFLP]. Genomic DNA was extracted from bull semen using phenol-chloroform method. A two-step PCR was conducted in order to amplify a 284 base-pair fragment of the target gene. Amplicons were digested by RsaI, HaeIII and BstyI restriction endonuclease enzymes. Digested fragments were electrophoresed on 8% polyacrylamide gel and visualized after silver staining. Seventeen BoLA-DRB3.2 alleles were identified with frequencies ranging from 1 to 21%. Sixteen alleles were similar to those reported previously and one was a new allele which has not been reported before. The frequencies of alleles BoLA-DRB3.2 *3, *8, *10, *11, *12, *13, *15, *16, *21, *22, *23, *24, *28, *51, *iaa, *ibb, *qbb were 2, 9, 2, 14, 1, 2, 4, 10, 1, 14, 5, 21, 6, 6, 1, 1, and 1%, respectively. The seven most frequent alleles [BoLA-DRB3.2 *8, *11, *16, *22, *24, *28, *51] accounted for 80% of alleles in the investigated population. This data indicate that the BoLADRB3.2 locus is highly polymorphic in Holstein bulls of Iran