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The PowerPlex Fusion 6C PCR™ amplification kit provides a strong discriminatory power for human identification. We have validated the kit with a reduced volume (12.5 µL) and as part of the validation we compared the efficiency of the polymerase chain reaction (PCR) prepared manually and on Hamilton Microlab® Autolys STAR Biorobot. Three years of casework data has been also included in the validation. Optimisation was carried out on different types of samples (blood, saliva, semen) and DNA was extracted robotically. Tests were conducted at two different cycle numbers (30;32), followed by analysis on both the Applied BiosystemsTM 3500 and 3500 xL Genetic Analyzer instruments (Applied Biosystems®, Foster City, CA, USA). When the PCR was prepared manually, no allele dropout was observed over 0.15 ng input DNA. Whereas when the PCR was prepared robotically, dropout already appeared at the level of 0.15 ng input DNA. In cases when increased cycle number was utilised, an increasing number of dropouts started to arise from 0.075 ng total input DNA. Despite the fact that robotically prepared PCR produced more missing alleles than the manually prepared PCR, using the optimal 0.5 ng input DNA, both methods proved to be reliable. Based on the results, our half-volume protocol is robust, and after three years of application it has proven to be effective with respect to a large number of casework samples. [ABSTRACT FROM AUTHOR]

Rapid diagnosis is key to containing viral outbreaks. However, for the current monkeypox outbreak the major deterrent to rapid testing is the requirement for higher biocontainment of potentially infectious monkeypox virus specimens. The current CDC guidelines require the DNA extraction process before PCR amplification to be performed under biosafety level 3 unless vaccinated personnel are performing assays. This increases the turn-around time and makes certain laboratories insufficiently equipped to handle specimens from patients with suspected monkeypox infection. We investigated the ability of five commercially available lysis buffers and heat for inactivation of monkeypox virus. We also optimized the use of monkeypox virus in Hologic® Panther Specimen Lysis Buffer for detection of virus in the Panther Fusion® Open Access System using published generic and clade specific monkeypox virus primers and probes. [ABSTRACT FROM AUTHOR]

Rapid diagnosis is key to containing viral outbreaks. However, for the current monkeypox outbreak the major deterrent to rapid testing is the requirement for higher biocontainment of potentially infectious monkeypox virus specimens. The current CDC guidelines require the DNA extraction process before PCR amplification to be performed under biosafety level 3 unless vaccinated personnel are performing assays. This increases the turn-around time and makes certain laboratories insufficiently equipped to handle specimens from patients with suspected monkeypox infection. We investigated the ability of five commercially available lysis buffers and heat for inactivation of monkeypox virus. We also optimized the use of monkeypox virus in Hologic® Panther Specimen Lysis Buffer for detection of virus in the Panther Fusion® Open Access System using published generic and clade specific monkeypox virus primers and probes.

Synovial fluid is important for the preoperative etiological diagnosis of suspected periprosthetic joint infection (PJI) or septic arthritis (SA). GENECUBE, an automated real‐time polymerase chain reaction (PCR) assay, was used to detect bacterial mecA (methicillin resistance) and was compared with microbiological cultures for preoperatively diagnosing PJI and SA in 74 patients suspected of these infections and thus earmarked for surgery. PJI and SA were diagnosed in 21 and 6 cases, respectively, using modified ICM 2018 diagnostic criteria. Microbiological cultures determined methicillin‐resistant staphylococcus (MRS) as the causative organism in six samples, which were all positive in the GENECUBE assay. Significantly also, the GENECUBE assay detected six MRS infections in culture‐negative but infection‐diagnosed patients, and in one inconclusive case, suggesting a higher sensitivity of this assay. Compared with microbiological culture, the sensitivity and specificity of the GENECUBE assay for mecAwas 100% and 92.2%, respectively. However, GENECUBE also produced invalid results in three cases, suggesting possible PCR inhibitors in the synovial fluid samples. We additionally validated the accuracy of pan‐bacterial real‐time PCR targeting 16S rRNA and other tests. Pan‐bacterial real‐time PCR was as effective as preoperative bacterial culture testing, although the α‐defensin assay had the highest sensitivity at 100%. Hence, fully automated real‐time PCR targeting of the bacterial mecA gene improves the etiological diagnosis of PJI and SA by reducing the testing time and lowering the false‐positive detection rates. A screening approach for α‐defensin followed by bacterial mecA gene testing in synovial fluids is therefore a more efficient method of preoperatively diagnosing PJI and SA. [ABSTRACT FROM AUTHOR]

Molecular detection of viral infections has the potential to improve microbial diagnostics, particularly with the emergence of rapid automated systems. We describe the design of the IDbox fully automated cassette-based system for nucleic acid extraction and real-time PCR amplification and perform a clinical evaluation for the diagnosis of genital herpes simplex infections. At optimal cutoff values determined by receiver-operator curves, the IDbox showed sensitivities of 94.9% (95% confidence interval [CI] 84.9–98.7%) and 97.0% (95% CI 88.5–99.5%) and specificities of 96.7% (95% CI 91.2–98.9%) and 97.3% (95% CI 91.9–99.3%) relative to herpes simplex virus culture and PCR, respectively. We discuss relevant design characteristics and approaches used for each step of the analytical process to enhance assay sensitivity and provide accurate results in the presence of potential cross-reactive organisms and interfering substances. [ABSTRACT FROM AUTHOR]

X-linked intellectual disability (XLID), also known as X-linked mental retardation, is a highly genetically heterogeneous condition for which mutations in >90 different genes have been identified. In this study, we used a custom-made sequencing array based on the Affymetrix 50k platform for mutation screening in 17 known XLID genes in patients from 135 families and found eight single-nucleotide changes that were absent in controls. For four mutations affecting ATRX (p.1761M>T), PQBP1 (p.155R>X) and SLC6A8 (p.390P>L and p.477S>L), we provide evidence for a functional involvement of these changes in the aetiology of intellectual disability. [ABSTRACT FROM AUTHOR]

Two mechanical lysis devices have been developed as compact, robust components to provide rapid sample preparation for nucleic acid diagnostic systems. One such component, known as the Micro Bead-BeaterTM (μBBTM, BBTM, Claremont BioSolutions, Upland, CA), is a compact device that is capable of ultra-rapid lysis (>90% lysis in 30s) of micro volumes (<80μL) of Bacillus spores in a continuous-flow format or in a disposable single-tube format. The μBB is also capable of processing much larger volumes of solutions containing spores or vegetative cells using a continuous-flow mode. A second mechanical lysis device designed as a disposable component is the microfluidic bead blender, which uses a small electric motor to spin vanes within the bead-laden solution. DNA quantification results using dsDNA-binding fluorescence dyes and real-time PCR are presented, comparing the lysis of Bacillus subtilis spores using the μBBTM with other well-known lysis techniques. Nanoscale imaging results obtained using scanning electron microscopy and transmission electron microscopy on B. subtilis spores lyzed using the μBBTM are also presented. [Copyright &y& Elsevier]

Introduction: To evaluate the automated cartridge-based PCR approach ARIES SARS-CoV-2 Assay targeting the ORF-sequence and the N-gene of SARS-CoV-2., Methods: In line with the suggestions by Rabenau and colleagues, the automated ARIES SARS-CoV-2 Assay was challenged with strongly positive samples, weakly positive samples and negative samples. Further, intra-assay and inter-assay precision as well as the limit-of-detection (lod) were defined with quantified target RNA and DNA. The Cepheid Xpert Xpress SARS-Cov-2 Assay was used as gold standard., Results: Concordance between the ARIES assay and the Cepheid assay was 100% for strongly positive samples and for negative samples, respectively. For weakly positive samples as confirmed applying the Cepheid assay, a relevant minority of 4 out of 15 samples (26.7%) went undetected by the ARIES assay. Intra- and inter-assay precision were satisfactory, while the lod was in the 103 DNA copies/reaction-range, in the 103 virus copies/reaction-range, or in the 103-104 free RNA copies/reaction-range in our hands., Conclusions: The automated ARIES assay shows comparable test characteristics as the Cepheid assay focusing on strongly positive and negative samples but a slightly reduced sensitivity with weakly positive samples. Decisions on diagnostic use should include considerations on the lod.