RESUMO
This article looks at storage factors influencing the stability of potential DNA calibration standards for use in quantitative polymerase chain reaction (PCR). Target sequences from the bacteria Campylobacter jejuni were cloned into a plasmid vector. Samples of these potential calibration standards were stored at +4, -20, and -80 degrees C as aqueous and lyophilized samples and were prepared as both single-use aliquots and multiple-use preparations. Results showed that the samples stored as single-use aqueous solutions at +4 degrees C and lyophilized samples stored at +4 and -20 degrees C were the most stable. Samples stored as frozen aqueous solutions at -20 degrees C were the least stable.
Assuntos
DNA/química , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Manejo de Espécimes/métodos , Calibragem , Campylobacter jejuni , DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Armazenamento de Medicamentos/métodos , Gastroenteropatias/microbiologia , Saúde Pública , Fatores de TempoRESUMO
Quantitative real-time PCR is one of the newer methods for measurement of the amount of nucleic material in biological systems. However, reliable measurement requires an appropriate estimation of uncertainty and this paper has developed the uncertainty budget associated with this procedure using as an example, data from a quantitative real-time PCR method for the enumeration of Campylobacter jejuni. This uncertainty is relatively large and for instance, a measured result of 151 units of DNA would have a 95% confidence interval of +/-84 units of DNA with the main sources of uncertainty being the measurement of the threshold cycle (Ct) value, the predicted DNA content of the unknown sample from the calibration line and the molar absorbance value for DNA.
Assuntos
Campylobacter jejuni/genética , Técnicas de Química Analítica/métodos , DNA Bacteriano/análise , Reação em Cadeia da Polimerase/métodos , Campylobacter jejuni/química , Técnicas de Química Analítica/normas , Reprodutibilidade dos Testes , Taq Polimerase/metabolismoRESUMO
The Tepnel Bio Kit for the detection of beef in cooked foods was assessed to determine its validity in demonstrating if food being imported into New Zealand contains beef material. The test suffered no interference from the presence of other common nonbovine species meats accepted as food within New Zealand and it detected beef in cooked samples of mixed meats when the proportion of beef in the mixture was >2 or >1%, depending on other meat species present. The documentation supplied with the kit indicates that the specific proteins it measures in cooked beef are stable to 130 degrees C. This was confirmed in the literature when the kit was used to test meat and bone meal cooked to at least 133 degrees C. However, our results showed these proteins to be much less stable when heated to elevated temperatures in moist food under pressure, and samples containing beef ceased to be positive by the immunoassay test after being autoclaved to 121 degrees C. This suggests that the test may not be able to detect even relatively high levels of beef in low-acid canned foods, which are normally retorted under pressure to approximately 121 degrees C.
