From Sample to Result – Workflow Solutions for Genotyping and Pathogen Detection

Editor's Notes

• #6: 1.) Proceedings of the National Academy of Sciences – reviewed that then microbial composition and gene content at these locations vary significantly from one person to the next. Consequently, it’s long been suspected that microbial communities found at certain body sites might have forensic utility. 2.) Sciences Advances today – westernization leads to reduction of diversity, to different compositions, and also to the activation of resistance genes in our bacteria. 3.) Nature Communications – found children who’ve taken certain antibiotics like azithromycin and clarithromycin experience long-lived shifts in their gut microbiome makeups, and metagenomic and other analysis of the intestinal microbiome of hundreds children review that macrolide use was linked with an increased risk of asthma and weight gain. 4.) Current Biology – gut microbiomes disproportionately affect some 11 Native American tribes. Microbiome population profiling – it matters when it comes to conditions such as obesity, type 2 diabetes, and autoimmune diseases. IBD – Inflammatory Bowel Disease, IBS – Irritable Bowel Sydrome。 5.) 2016 Personalized Medicine World Conference in Silicon Valley – show growing understanding of the interplay between the microbiome and human disease has opened up new opps. For theapeutic interventions aimed at modifying disrupted or dysfunctional microbiota
• #12: QIAamp DNA Stool Mini Kit – Manual Silica membrane-based technology No organic extraction or alcohol precipitation Complete removal of contaminants and inhibitors Sample type: fresh or frozen human stool or others sample types with high concentrations of PCR inhibitors Yields: 10–30 µg Time: 50 minutes Elution volume: 200 µl
• #31: Tritrichomonas foetus is a single celled flagellated protozoan parasite that is known to be a pathogen of the bovine reproductive tract as well as the intestinal tract of cats. In cattle, the organism is transmitted to the female vagina and uterus from the foreskin of the bull where the parasite is known to reside. It causes infertility, and, at times, has caused spontaneous abortions in the first trimester. Cases of infection in cattle have declined due to the use of artificial insemination. In the last ten years, there have been reports of Tritrichomonas foetus in the feces of young cats that have diarrhea[1] and live in households with multiple cats.
• #32: Calf scours is caused by bacterial, viral and sometimes even by parasitic infections. Newborn dairy and beef animals are especially susceptible to calf scours because their immunity systems are not fully developed. Severe fluid loss due to calf scours results in dehydration and often leads to death. Unfortunately, even animals that survive calf scours often remain weak and perform poorly throughout their lives. Common Names Calf scours is also called calf diarrhea, or neo-natal diarrhea. Many consider calf scours as not a specific disease with a specific cause, but actually as a clinical sign of a complex disease. Whatever the truth may be, Calf scours is the primary cause of death in calves from 2 to 30 days of age. Causative Organism According to a leading research organization, the causative organisms of calf scours may be Coronavirus, Rotavirus, K99 E. coli bacteria or Clostridium perfringens Type C. 
• #34: Bovine respiratory disease (BRD) is the most common and costly disease affecting beef cattle in the world.[1] It is a complex, bacterial infection that causes pneumonia in calves and can possibly be fatal. The infection is usually a sum of three codependent factors: Stress, an underlying viral infection, and a new bacterial infection.[2] The diagnosis of the disease is complex since there are multiple possible causes.[3] The disease manifests itself most often in calves within four weeks of weaning, when calves are sorted and often sold to different farms. This gives it a common nickname, "Shipping Fever." It is not known whether the stress itself, commingling, or travel conditions are at most to blame, and while studies have identified general stressing factors like transport and cold weather conditions, there is still no conclusive evidence on more specific factors (e.g. distance, transport mode, temperature, or temperature volatility.
• #38: P.Multocida are involved in most costly to North American cattle industry. So called: “shipping fever pneumonia”
• #41: Current customer Workflow: Extraction: Mo Bio PowerSoil DNA Isolation Kit (manually) Standard culture method on agar to ID micro- organism population and profiling (manual) Platting method currently working to discriminate top 5 organisms… to what extend, able to meet FDA requirements? What is the method used in Trial II-B currently? MoBio PowerFecal, Powersoil DNA Isolation kits  Agilent MX3005P for q-PCR (4 channels; manual). Samples: 16 DNA samples, 100ul @ greater than 15 ng/ul concentration. Samples will include reference samples from > three individuals extracted from Feces and soil with Powerfecal and Powersoil kits. 48- 64 human fecal and soil samples from multiple subjects. All of which will be QC’ed and a comparable potion of them will be assayed.
• #53: RotorGene and HRM Type-it Chemistry: Quantification on PMA treatments -- Have created new protocols; demonstrated real-time PCR by Roter-GeneQ and HRM kit can detect down to ~1% Live PMA treated cells with good sensitivity, correlation (Slope= -3.22; Eff. = 100%; R2= 0.9983), comparable assay time, and linearity that is far better than the data created by “prepMan Ultra Kit” and Agilent MX3005P (Slope= -4.32; Eff. = 70.5%) HRM donor sample profiling-- Have demonstrated that the combination of ScreenClust software and Rotor-Gene Q instrument is capable of separating multiple sub-clusters from reference population of 12 healthy subjects. Full Automation – A “Walk away”, cost-saving solution: Have created customized protocols, and demonstrated QIAcube High Throughput (HT) with QIAamp DNA virus kit can extract human fecal sample from clinical treatments with better consistency. The nanodrop data have demonstrated good concentration ranges, yield, and purity and salt ratios. The real-time PCR data have shown good Ct range compare to previously extracted customer DNAs extracted from MO Bio “PowerFecal DNA Isolation kit”. QIAgility liquid handler/ assay setup instrument is ideal for fully automated assay setup on 72-Disc and 100-Disc rings. Data from previous customers (data not shown here) had demonstrated 28-32 min/ 100 samples capacity with greater consistency, time saving, and better %CV compare to manual setup.