Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

Streptococcus Group B (GBS) colonization in pregnant women is the most important risk factor for newborn disease due to vertical transmission during delivery. GBS colonization during pregnancy has been implicated as a leading cause of perinatal infections. Traditionally, pregnant women are screened for GBS between 35 and 37 weeks of gestation. However, antenatal culture-based screening yields no information on GBS colonization status and offers low predictive value for GBS colonization at delivery. Numerous assays have been evaluated for GBS screening in an attempt to validate a fast and efficient method. The aim of this study was to compare bacteria isolation by culture and two qPCR techniques, targeting sip and cfb genes, respectively, for detecting colonizing GBS. Cultures – the gold-standard technique, a previous qPCR technique targeting the sip gene, and a new proposed qPCR assay targeting the cfb gene were evaluated as diagnostic tools on 320 samples. Considering cultures as the gold standard, the evaluated qPCR method detected 75 out of 78 samples, representing a sensitivity of 93.58% (95% confidence interval (CI), 90.89–96.27) and specificity of 94.62% (95% CI, 91.78–97.46). However, an additional analysis was performed for true positives that included not only samples showing positives by culture but samples showing positive for both qPCR assays. The sensitivity and specificity were recalculated including these discrepant samples and a total of 89 samples were considered as positive, giving a prevalence of 27.81%. With this new analysis, the qPCR targeting the cfb gene showed a sensitivity of 95.5% (95% CI, 88.65–98.59) and specificity of 99.13% (95% CI, 96.69–99.97). The new qPCR method is a sensitive and specific assay for detecting GBS colonization and represents a valuable tool for identifying candidates for intrapartum antibiotic prophylaxis. Cultures should be retained as the reference and the routine technique because of its specificity and cost analysis ratio, but it would be convenient to introduce PCR techniques to check negative culture samples or when an urgent detection is required to reduce risk of infection among infants.

Objective: Peripheral blood is the most promising source of RNA biomarkers for diagnostic and epidemiological studies, because the presence of disease and prognostic information is reflected in the gene expression pattern. Quality RNA is used by a number of different downstream applications, so the selection of the most appropriate RNA stabilization and purification method is important. We have analyzed the RNA purified from 300 blood samples from 25 donors processed by two technicians using three methodologies with Tempus and PaxGene tubes. Results: The best quality sample results were obtained with the Tempus Spin RNA Isolation Kit and the PaxGene Blood miRNA Kit, although larger amounts of RNA were obtained with the Tempus Spin RNA Isolation Kit. Lower Cq values were observed for RNA and miRNA genes in samples that were tested with PaxGene Blood miRNA Kit and Tempus Spin RNA Isolation Kit respectively. We identify the Tempus Spin RNA Isolation Kit as the most robust methodology, whilst the MagMax for Stabilized Blood Tubes RNA Isolation Kit showed the most instability. For biobanks, which process a large cohort and conduct epidemiological studies, the Tempus Spin RNA Isolation Kit is the most appropriate methodology. The study demonstrates the robustness of real-life procedures.

In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of Mycoplasma in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor^®GeM qEP and Plasmotest^® kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded.

Cell lines are a widely used pre-clinical models for biomedical research. The accessibility and the relative simplicity of facilities necessary for the use of cell lines, along with the large number of potential applications, encourage many researchers to choose this model. However, the access to cell lines from a non-confident source or through the interlaboratory exchange results in uncontrollable cell lines of uncertain quality. Furthermore, the possibility of using cell lines as an endless resource through multiple passages can contribute to this uncontrolled scenario, the main consequence of which is the lack of reproducibility between the research results. Different initiatives have emerged to promote the best practices regarding the use of cell lines and minimize the effect on the scientific results reported, including comprehensive quality control in the frame of Good Cell Culture Practice (GCCP). Cell Banks, research infrastructures for the professional distribution of biological material of high and known quality and origin, are committed with these initiatives. Many of the quality controls used to test different attributes of cell lines are based on DNA. This review describes quality control protocols of cell lines whose target molecule is DNA, and details the scope or purpose and their corresponding functionality.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus, is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) −7 to 42, from pigs (n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples (n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples (n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs (n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.