Choosing the best sample prep method for your detection assay
Nucleic acid-based methods are used to detect viral and bacterial pathogens in a wide variety of sample types, including clinical research and environmental samples. The accuracy and efficiency of detection methods such as real-time quantitative PCR (RT-qPCR) analysis and PCR-based genotyping are often limited by difficulty in purifying high-quality RNA and DNA from samples to be analyzed. It is important to choose a purification method suitable for a given sample type and organism, simple and rapid enough to avoid significantly increasing the time necessary to complete a detection assay, and appropriate for the amount of sample and number of samples being processed.
Silica membrane- and magnetic bead-based technologies allow for the rapid purification of viral and microbial nucleic acids at various throughputs using either manual or automated processing. These technologies have been incorporated into versatile kits suitable for a wide range of samples, as well as specialized kits designed for specific purification formats and sample types such as stool, soil, and water samples that contain hard-to-lyse microorganisms. Alternatively, some RT-qPCR methods are well-suited for small-scale detection from crude extracts, eliminating the need for prior DNA or RNA isolation altogether.
Please see the following relevant examples and view our selection guide to find the best nucleic acid purification kit for your application.
Please note that Macherey-Nagel nucleic acid purification products are only available from Takara Bio in North America, India, and Japan.
Preparing clinical research samples for viral detection
Takara Bio offers both silica membrane- and magnetic bead-based technologies for the purification of viral nucleic acids from cell-free bodily fluids such as serum or plasma, blood, homogenized tissue and stool sample suspensions, and swab washes with the NucleoSpin RNA Virus and NucleoMag Pathogen kits. Both kits incorporate simple, rapid lysis procedures that enable efficient isolation of nucleic acids from RNA and DNA viruses, while NucleoMag Pathogen provides added versatility for extraction of bacterial DNA. Extracted nucleic acids are then bound to a silica membrane or paramagnetic beads, respectively, subjected to sequential wash steps, and eluted under low-salt conditions. While NucleoSpin silica membrane technology is available in both single-prep and multi-prep formats (NucleoSpin 8/96 Virus kits) for varying throughput demands, the NucleoMag Pathogen kit has been developed primarily for high-throughput processing on many common automation platforms. The features of the three kits are compared in the table below.
| NucleoSpin RNA Virus | NucleoSpin 8/96 Virus* | NucleoMag Pathogen | |
|---|---|---|---|
| Technology | Silica membrane | Silica membrane | Magnetic beads |
| Format | Mini spin columns | 8-well strips/96-well plates | Highly reactive superparamagnetic beads |
| Processing | Small-scale manual processing | High-throughput manual or automated processing* | High-throughput manual or automated processing |
| Starting material | 150 µl | 150 µl | <200 µl (prep is fully scalable) |
| Fragment size | 100 bp–30 kb | 100 bp–30 kb | 300 bp–~ 50 kb |
| Elution volume | 50 µl | 70–100 µl | 50–100 µl |
| Preparation time | 30 min/4–6 preps | 60 min/6 strips 60 min/plate |
45–120 min/96 preps (e.g., 45 min for KingFisher) |
*NucleoSpin 8 Virus and NucleoSpin 96 Virus are formatted for manual sample processing by centrifuge or vacuum. NucleoSpin 8/96 Virus Core kits exclude components required for manual use and are intended primarily for automated sample processing under vacuum.
These kits yield high-quality viral nucleic acids that can be used in detection assays for a wide range of sample types. They enable efficient isolation of viral nucleic acids from clinical research samples such as nasopharyngeal and oropharyngeal swabs, sputum, and other bodily fluids. We provide guidelines for nucleic acid isolation from these types of samples that are suitable for SARS-CoV-2 research applications. The NucleoSpin RNA Virus kits have also been shown to work with HCV, HIV, HPV, bird flu virus, and bluetongue virus, and may be used to purify HBV DNA with the addition of a Proteinase K digestion step. The NucleoMag Pathogen kit can also purify nucleic acids from a wide array of viruses and bacteria. It was shown to outperform a competitor in enabling more sensitive PCR-based detection of five common pathogens (norovirus, Clostridium difficile, astrovirus, adenovirus, and rotavirus) after both kits were used to extract nucleic acids from a stool sample (Figure 1).
Figure 1. Sensitivity screening for pathogen detection in human fecal samples. Dilution series of 10-3–10-4 of fecal samples were prepared for sensitivity screening using three replicates. Nucleic acid extraction was performed with the NucleoMag Pathogen kit and the competitor kit "R". PCR analysis was performed using the RIDA GENE Viral Stool Panel I (R-Biopharm) and RealStar Clostridium difficile PCR Kit 1.0 (Altona Diagnostics).
Isolation of microbial DNA and RNA from environmental samples
Takara Bio also offers specialized kits that are effective for isolating viral nucleic acids from environmental samples such as water, air, and soil. The magnetic bead-based NucleoMag DNA/RNA Water kit enables efficient, high-throughput isolation of DNA and RNA from diverse water and air samples. The protocol incorporates ceramic bead homogenization for optimal lysis and efficiently removes inhibitory compounds to ensure optimal performance of downstream assays. This versatile kit was shown to yield microbial DNA that could be efficiently detected from a selection of various water and air samples using a PCR assay (Figure 2).
Figure 2. Efficient detection of microbial DNA from different water and air samples. DNA was extracted from various filtered water and air samples using the NucleoMag DNA/RNA Water kit and analyzed by PCR. Microbial DNA could be efficiently measured for all of the samples, demonstrating the versatility of the NucleoMag DNA/RNA Water kit.
Other kits, such as NucleoBond RNA Soil and NucleoSpin RNA Stool, are useful for isolating viral nucleic acids from water samples. We provide general recommendations for isolating viral nucleic acids such as SARS-CoV-2 RNA from water samples using these kits.
Direct detection of pathogens from crude extracts
Direct RT-qPCR may be used to detect pathogens directly from a variety of crude biological samples, eliminating the need for RNA purification. Our PrimeDirect Probe RT-qPCR Mix can be used for direct RT-qPCR detection because it is highly resistant to various PCR inhibitors such as heparin (blood) and humic acid (soil). This master mix, which is used to perform a highly sensitive fluorescent probe-based assay, enables direct one-step detection in a single tube, significantly reducing hands-on time and risk of contamination. An H1N1 assay performed using this mix was shown to be highly sensitive, detecting as few as 20 copies of the virus in nasal cavity swab samples and saliva samples and two copies from mouth samples (Figure 3).
Figure 3. PrimeDirect Probe RT-qPCR Mix enabled the detection of viral RNA directly from biological samples. The detection sensitivity from nasal cavity swab samples and saliva samples was 20 copies, and the sensitivity from mouth swab samples was two copies.
Takara Bio also offers the Direct One-Step RT-qPCR Mix for SARS-CoV-2, a specialized one-step RT-qPCR master mix that allows direct detection of SARS-CoV-2 from unpurified saliva samples in a simple, rapid protocol—just mix the sample and pretreatment reagent, heat treat, and then combine with the RT-qPCR master mix. The entire protocol, from pretreatment through detection, takes less than one hour and includes guidelines for multiplexed SARS-CoV-2 detection. This master mix, which is based on the exceptionally sensitive One Step PrimeScript III RT-qPCR Mix, was shown to provide reliable detection of SARS-CoV-2 RNA in crude saliva samples.
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