Technical note: Development of DNA quantitation and STR typing systems for Panthera tigris species determination and individual identification in forensic casework

The aim of this technical note is to provide an overview of methodical approaches used to develop molecular systems for species determination/DNA quantification called Ptig Qplex and individual identification called Ptig STRplex of Panthera tigris samples. Both systems will help to combat the illegal trade of endangered species and create a worldwide shared database of DNA profiles.

Illumina DNA Prep (M) Tagmentation Library Preparation for use on an Illumina MiSeq Sequencer v2

This procedure outlines the protocol for whole genome sequencing of bacterial organisms using the Illumina DNA Prep library preparation kit for sequencing on an Illumina MiSeq sequencer. This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network. Complete in order: 1. DNA Extraction (Manual DNA Extraction or Automated DNA Extraction using the Qiacube) Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing 2. DNA Quantitation Quantitation of extracted DNA using the Qubit Flourometer 3. Library Preparation for WGS (Included SOP or Library Preparation using Illumina Nextera XT ) Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex) 4. Sequencing using Illumina MiSeq 5. Data Quality Checks and NCBI Submission

Comparison of the Amplisure HBV Quantitative Kit with the Qiagen Artus HBV QS-RGQ Assay for Quantifying Viral DNA in Plasma Samples of Monitoring Cases

<b><i>Background:</i></b> Monitoring of hepatitis B virus (HBV) viral load has become an essential phase in the treatment of HBV. There are many commercial assays available for HBV viral load quantification. In this study, we have evaluated the performance characteristics of Amplisure® HBV Kit in comparison with the Qiagen artus HBV QS-RGQ kit for HBV DNA quantitation. <b><i>Methods:</i></b> Comparison of 2 methods was carried out on 200 clinical samples, 150 HBV DNA positive and 50 HBV DNA negative, by a reference method. Results obtained with Amplisure® HBV Kit (Amplisure HBV) were compared using the Qiagen artus HBV QS-RGQ assay results as the comparator method. <b><i>Result:</i></b> The overall performance of the Amplisure HBV compared with the comparator method shows positive and negative clinical agreement of 100 and 76%, respectively. Among the 12 qualitative discrepant samples, all positive with Amplisure HBV were sequenced and 10 were below comparator method’s LOD. For 5 weak positives (−0.22 to 0.98 log IU/mL), the sequencing failed. The 7 other positives (0.48 to 1.89 log IU/mL) were confirmed positive by sequencing. Quantitative comparison gave an <i>r</i>² of 0.967 with a mean log difference of 0.09 log₁₀ IU/mL. <b><i>Conclusion:</i></b> This study shows that Amplisure® HBV Quantitative Kit shows comparable performance with artus HBV QS-RGQ assays and can be useful in management and therapeutic monitoring of HBV in a clinical practice.

Revisiting soil bacterial counting methods: Optimal soil storage and pretreatment methods and comparison of culture-dependent and -independent methods

Although a number of different methods have been used to quantify soil bacteria, identifying the optimal method(s) for soil bacterial abundance is still in question. No single method exists for undertaking an absolute microbial count using culture-dependent methods (CDMs) or even culture-independent methods (CIMs). This study investigated soil storage and pretreatment methods for optimal bacterial counts. Appropriate storage temperature (4°C) and optimal pretreatment methods (sonication time for 3 min and centrifugation at 1400 g) were necessary to preserve bacterial cell viability and eliminate interference from soil particles. To better estimate soil bacterial numbers under various cellular state and respiration, this study also evaluated three CDMs (i.e., colony forming unit, spotting, and most probable number (MPN) and three CIMs (i.e., flow cytometry (FCM), epifluorescence microscopy (EM) count, and DNA quantitation). Each counting method was tested using 72 soil samples collected from a local arable farm site at three different depths (i.e., 10–20, 90–100, and 180–190 cm). Among all CDMs, MPN was found to be rapid, simple, and reliable. However, the number of bacteria quantified by MPN was 1–2 orders lower than that quantified by CIMs, likely due to the inability of MPN to count anaerobic bacteria. The DNA quantitation method appeared to overestimate soil bacterial numbers, which may be attributed to DNA from dead bacteria and free DNA in the soil matrix. FCM was found to be ineffective in counting soil bacteria as it was difficult to separate the bacterial cells from the soil particles. Dyes used in FCM stained the bacterial DNA and clay particles. The EM count was deemed a highly effective method as it provided information on soil mineral particles, live bacteria, and dead bacteria; however, it was a time-consuming and labor-intensive process. Combining both types of methods was considered the best approach to acquire better information on the characteristics of indigenous soil microorganisms (aerobic versus anaerobic, live versus dead).

A novel method for removing polyethyleneimine from biopharmaceutical samples: improving assay sensitivity of residual DNA qPCR

Polyethyleneimine (PEI) is a flocculent that is widely used in the downstream purification of monoclonal antibodies. It is an in-process residual that is carried through the drug purification process and strongly inhibits residual DNA quantitation by real-time quantitative PCR assay. Very high sample dilutions (e.g., 1:10,000) can overcome the interference of PEI, but at the cost of DNA assay sensitivity. Diluting samples poses a significant risk to the assay sensitivity needed to satisfy regulatory requirements on the quantitation of residual genomic DNA present per dose (i.e., 10 ng/dose). Removing PEI while retaining DNA, by the use of sodium dodecyl sulfate, heparin and/or sarkosyl can overcome the interference of PEI and allow a more accurate quantitation of residual DNA.

UV SPECTROPHOTOMETER: PRACTICAL CONSIDERATIONS

This article deals with UV Spectroscopy its application (practical) and its mode. There are various modes of UV spectrophotometer namely photometric, spectrum, quantitation, kinetics, time scan, multi-component and bio-method. Photometric measures absorbance or % transmittance of a sample at arbitrary wavelengths. Spectrum Scans a wavelength range to measure the absorbance and % transmittance of a sample as a function of wavelength. Single beam energy measurement can also be performed. Data processing such as peak detection, smoothing, and mathematical calculation may be applied to the measured spectrum. Quantitation creates a calibration curve from a standard sample and quantitates an unknown sample. Kinetics Calculates enzyme activity from the time dependent change in absorbance. Time scan This function is used to measure the change in the rate of absorbance, transmittance, or energy in the fixed wavelength. Multi-component Enables samples with up to 8 constituent components to be measured and quantitated. In the Bio-method Mode, DNA Quantitation and Protein measurement are done. These modes are used for different analysis.