MATERIALS AND METHODS: The EGFR intron 1 polymorphism was analysed in three distinct healthy Asian subjects, namely, Chinese (N = 96), Malays (N = 98) and Indians (N = 100). Comparative genomic hybridisation was performed to investigate for changes in DNA copy number in relation to the polymorphic CA dinucleotide repeats in breast tumor tissues (N = 22).
RESULTS: The frequency of short alleles with 14 and 15 CA repeats were most common in the Asian populations and significantly higher than those reported for Caucasians. The frequency of 20 CA repeats was 5%, almost 13-fold lower than previous reports. EGFR amplifications were detected in 23% and 11% of breast tumor tissues harboring short and long CA repeats, respectively.
CONCLUSION: Our results show that the frequency of alleles encoding for short CA dinucleotide repeats is common in Asian populations. EGFR expression and amplification levels were also higher in Asian breast tumor tissues with short CA dinucleotide repeats. These findings suggest that the EGFR intron 1 polymorphism may influence response to treatment with tyrosine kinase inhibitors in breast cancer patients and further studies are warranted.
DESIGN: Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks.
RESULTS: Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3.
CONCLUSION: Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia.
RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases.
CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.