Aims: To demonstrate that produce rinsates used for RT‐qPCR detection of foodborne viruses may cause significant PCR inhibition and propose a means to reduce its impact on sensitivity. Methods and Results: Here, it is shown that rinsing and concentration from spinach and precut lettuce have the potential to generate RNA extracts that are inhibitory to RT‐qPCRs assembled from commercial kits for the detection of norovirus GII (NoV GII), hepatitis A virus (HAV), hepatitis E virus (HEV), rotavirus (RV) and feline calicivirus (FCV) as sample process control. It is further shown that the addition of bovine serum albumin (BSA) to those reactions restored a positive signal in all cases. The effect of BSA was dependent upon the primer/probe combination. Moreover, two of the detection systems (FCV and HAV) strongly benefited from the addition of BSA even in the absence of PCR inhibitors. Conclusions: BSA was shown to restore positive signals in five different RT‐qPCR systems that were otherwise completely inhibited by produce rinsate extracts. It is therefore suggested to consider the addition of BSA to RT‐qPCRs for the detection of foodborne viruses when inhibition is observed. Significance and Impact of the Study: This study clearly demonstrates the potency of PCR inhibitors generated during routine virus concentration from produce and that it can be alleviated by the addition of BSA to the RT‐qPCRs. Although used elsewhere, the addition of BSA to PCRs is not a common practice in this growing field of research.
BackgroundCounterfeit and unapproved medicines are inherently dangerous and can cause patient injury due to ineffectiveness, chemical or biological contamination, or wrong dosage. Growth of the counterfeit medical market in developed countries is mainly attributable to life-style drugs, which are used in the treatment of non-life-threatening and non-painful conditions, such as slimming pills, cosmetic-related pharmaceuticals, and drugs for sexual enhancement. One of the main tasks of health authorities is to identify the exact active pharmaceutical ingredients (APIs) in confiscated drugs, because wrong API compounds, wrong concentrations, and/or the presence of chemical contaminants are the main risks associated with counterfeit medicines. Serious danger may also arise from microbiological contamination. We therefore performed a market surveillance study focused on the microbial burden in counterfeit and unapproved medicines.MethodsCounterfeit and unapproved medicines confiscated in Canada and Austria and controls from the legal market were examined for microbial contaminations according to the US and European pharmacopoeia guidelines. The microbiological load of illegal and legitimate samples was statistically compared with the Wilcoxon rank-sum test.ResultsMicrobial cultivable contaminations in counterfeit and unapproved phosphodiesterase type 5 inhibitors were significantly higher than in products from the legal medicines market (p < 0.0001). Contamination levels exceeding the USP and EP limits were seen in 23% of the tested illegal samples in Canada. Additionally, microbiological contaminations above the pharmacopoeial limits were detected in an anabolic steroid and an herbal medicinal product in Austria (6% of illegal products tested).ConclusionsOur results show that counterfeit and unapproved pharmaceuticals are not manufactured under the same hygienic conditions as legitimate products. The microbiological contamination of illegal medicinal products often exceeds USP and EP limits, representing a potential threat to consumer health.
An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal withinand between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H202) and hydrogen donor [2,2'-azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both withinand between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.
Six foods representing a variety of food products were analyzed by the Assurance Listeria polyclonal enzyme immunoassay (EIA) and by either the Bacteriological Analytical Manual or the U.S. Department of Agriculture culture method for detecting Listeria monocytogenes and related Listeria species. Samples of each food type, at each inoculation level, were analyzed simultaneously by both methods. A total of 19 laboratories representing federal government agencies and private industry in the United States and Canada participated. Food types were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots of green beans, which were naturally contaminated. During this study, 1764 samples and controls were analyzed and confirmed, of which 492 were positive and 947 were negative by both methods. There were 159 samples that were positive by culture method but negative by the EIA and 188 that were negative by culture method but positive by EIA. Twenty-two samples were negative by EIA and by culture method but confirmed positive when Assurance selective enrichment broths were subcultured to selective agar. The Assurance polyclonal EIA for detecting L. monocytogenes and related Listeria species in foods has been adopted first action by AOAC INTERNATIONAL.
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