[Detection of Mycoplasma salivarium contamination in cell culture using PCR method]

Mycoplasma salivarium [M. salivarium] is one of the most common contaminants present in cell culture laboratories that cause undesirable effects on cell cultures. Thus, the identification and rapid diagnosis in controlling and prevention of this contaminant are important. The aim of this study is the detection of Mycoplasma salivarium contamination in cell culture using polymerase chain reaction [PCR] method. A 16S rRNA-based Mycoplasma genus and specific primer PCR method for M. salivarium was developed. The sensitivity and specificity of this method were determined. The PCR test was used after we extracted DNA from the cultured isolates. A total of 62 cell culture samples were sent to the Mycoplasma Reference Laboratory at Razi Institute, Karaj, Iran for detection of Mycoplasma contamination. A total of 42 [67.75%] out of 62 samples scored positive according to the Mycoplasma genus. From these 42 samples, 15 [35.72%] reacted positively with a clear band of 434 bp in the M. salivarium-specific PCR method. Due to the high percentage of M. salivarium contamination in cell cultures, we recommend aseptic conditions be used in the laboratory when working with cell cultures. The PCR method is a suitable and valuable tool for the detection of M. salivarium contamination in cell cultures with appropriate and specific primers. This PCR method can be processed in less than one day

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

Sequence analysis and phylogenetic study of hemagglutinin gene of H9 N2 subtype of avian influenza virus isolated during 1998-2002 in Iran

Sequence analysis and phylogenetic study of hemagglutinin [HA] gene of H9N2 subtype of avian influenza virus isolates [outbreaks of 1998-2002] in Tehran province [Iran] were studied. Two sets of forward and reverse primers in highly conserved regions, based on sequences of HA gene in Genbank, were designed. PCR products of a 430-bp fragment of 16 isolates were sequenced and then were aligned with the reported sequences in Genbank. Nucleotide sequence comparisons of HA gene from Iranian isolates showed 97-99% identity within the group, and 98% homology with the two isolates [A/Parakeet/Narita/92A/98 [H9N2]] and [A/Parakeet/Chiba/1/97 [H9N2]] from Pakistani parakeets imported to Japan. On the basis of phylogenetic evidence, it is proposed that the emergence of H9N2 avian influenza infection in Iran originated in Pakistan, and it was due to low quarantine measures in the international boundaries. Due to the high percentage of H9N2 homology isolates of Iran with other isolates, namley A/quail/HongKong/G1, in Genbank and based on published reports for high similarity with infecting human H5N1 isolates, it seems that the potential of Iranian avian influenza isolates to infect human should be considered