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NadPure Microbial Pathogens RNA & DNA Kit

Catalog Number:

#1010301
#1010302

NadPure Microbial Pathogens RNA & DNA Kit enables the isolation and puriﬁcation of high-quality microbial RNA and DNA from a variety of clinical samples. The extraction process is safe, non-toxic, and easy to perform, without the use of hazardous reagents such as phenol or chloroform. While ensuring eﬃcient pathogen nucleic acid extraction, the kit eﬀectively removes proteins, inorganic salts, and other impurities.

The puriﬁed nucleic acids can be directly used for a variety of downstream applications, including PCR, qPCR, RT-PCR, and the library preparation of metagenomic next-generation sequencing (mNGS), targeted NGS (tNGS), and RNA sequencing (RNA-Seq).

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Feature

Broad Sample Compatibility: Supports the extraction of pathogen RNA and DNA from a wide range of clinical specimens, including blood, swabs, respiratory specimens, sterile body fluids, saliva, tissue, stool, and microbial cultures, meeting the testing needs of infectious specimens from diverse sources.

Combined Lysis for Enhanced Nucleic Acid Release: Combines mechanical disruption and chemical lysis to promote nucleic acid release from pathogens with different structural characteristics. It improves lysis efficiency for pathogens with thick cell walls while maintaining the integrity of low-abundance RNA viral nucleic acids.

Effective Impurity Removal and Efficient Recovery: Employs high-performance magnetic beads and a specially optimized buffer system to maximize the removal of impurities, such as proteins and salts, as well as potential inhibitors, enabling efficient recovery and purification of RNA and DNA.

High-Quality Nucleic Acids for Reliable Testing: Provides sufficient quantities of high-purity, high-integrity mixed RNA and DNA as high-quality templates for downstream molecular testing, helping ensure the accuracy and reliability of test results.

Strict Contamination Monitoring to Minimize Interference: Monitors exogenous microbial background contamination throughout the entire workflow, helping identify and exclude false-positive pathogen signals associated with background contamination, reducing the risk of pathogen misidentification and enhancing the reliability of test reports.

Simple Operation and Automation Compatibility: Based on magnetic bead separation technology, the kit features a simple workflow with low equipment dependency. It can be seamlessly integrated with fully automated nucleic acid extraction systems, supporting ultra-highthroughput parallel sample processing.

Workflow

Performance

Broad Compatibility with Clinical Infectious Samples from Diverse Sources

Figure 1. Co-Extraction performance of the NadPure Microbial Pathogens RNA & DNA Kit across different sample types. A. Yield of mixed RNA & DNA;B. Nucleic acid purity; C. Agarose gel electrophoresis of the extracted nucleic acids.
Note: BALF: bronchoalveolar lavage fluid; M: DNA Marker.

Enhanced Lysis Promotes Efficient Nucleic Acid Release from Hard-to-Lyse Pathogens

Figure 2. Enhanced lysis with the NadPure Microbial Pathogens RNA & DNA Kit promotes nucleic acid release from hard-to-lyse pathogens under low-pathogen-load conditions.A. S. aureus; B. A. fumigatus. The NadPure Microbial Pathogens RNA & DNA Kit (NadPure), Vendor A, and Vendor B were used to co-extract RNA and DNA from microbial reference materials containing S. aureus and A. fumigatus at known viability levels, following serial dilutions of 20-fold, 40-fold, 80-fold, and 160-fold. Subsequently, 2 μL of each nucleic acid extract was used for qPCR analysis.

Efficient, High-Quality Extraction Enables Sensitive Detection of Multiple Pathogens

Figure 3. Comparison of nucleic acid extraction performance across multiple pathogens in simulated clinical samples using different magnetic bead-based co-extraction kits.A. Plasma; B. Swab; C. Sputum. Simulated clinical samples were prepared using serially diluted microbial reference materials, including S. aureus, A. fumigatus, and Influenza A virus H9N2, with known viability levels and/or copy numbers. Specifically, 5 μL of microbial reference materials diluted 10-fold (High), 40-fold (Medium), and 160-fold (Low) were separately added to plasma, swab, and sputum samples. NadPure, Vendor A, and Vendor B werethen used to co-extract RNA and DNA from the simulated samples. Subsequently, 2 μL of each nucleic acid extract was used for qPCR analysis.

Whole Process Monitoring of Exogenous Background Microbial Contamination

Table 1. Evaluation using an in-house database demonstrates low levels of residual background microbial contamination in the NadPure Microbial Pathogens RNA & DNA Kit.

10 ng Human Genomic DNA Standard (Promega, G1521) was added to 400 μL of Nuclease Free Water to prepare simulated negative control samples. The NadPure Microbial Pathogens RNA & DNA Kit was used to co-extract RNA and DNA from the simulated samples. The resulting nucleic acids were then used for metagenomic sequencing library preparation with the NadPrep EZ RNA & DNA Library Co-Preparation Kit. Sequencing was performed on the NovaSeq 6000, PE150, generating 1 Gb of data.

Reliable Performance in Addressing the Challenges of Complex Infection Samples

Figure 4. Comparison of tNGS detection performance for different magnetic bead-based co-extraction kits using simulated complex clinical sample.Simulated complex clinical samples were prepared using serially diluted microbial reference materials, including plasma, swab, and sputum samples, with three independent samples for each sample type. The NadPure Microbial Pathogens RNA & DNA Kit and Vendor A were used to co-extract RNA and DNA from the simulated samples. The extracted nucleic acids were then used for pre-library preparation with the NadPrep EZ RNA & DNA Library Co-Preparation Kit, followed by hybrid capture using the μCaler HyperMix Hybrid Capture Kit (for Illumina®) and the μCaler RIA Panel v1.0. Sequencing was performed on the NovaSeq 6000, PE150.

Note: The microbial reference materials include S. enterica, H. influenzae, K. pneumoniae, P. aeruginosa, E. coli, S. pneumoniae, S. aureus, A. baumannii, E. faecium, Y. enterocolitica, A. fumigatus, A. niger, C. albicans, and Influenza A virus H9N2. RPM stands for reads per million mapped reads.

For research use only. Not for use in diagnostic procedures.

Package	Color of Cap Tube	Component	Volume 1010301 (24 rxn)	Volume 1010302 (96 rxn)
Box1	/	Lysis & Grinding Tube	24 unit	96 unit
	/	Patho-Lysis Buffer	5 mL	19.5 mL
	/	Patho-Binding Buffer	6 mL	24 mL
		Patho-Magnetic Beads	720 μL	2×1,440 μL
	/	Patho-Wash Buffer 1	25.5 mL	105 mL
	/	Patho-Wash Buffer 2	8 mL	32 mL
	/	Elution Buffer	2.5 mL	10 mL
Box2	or /	Proteinase K	960 μL	4 mL

For research use only. Not for use in diagnostic procedures.

Can the NadPure Microbial Pathogens RNA & DNA Kit be used to extract only RNA or DNA?

The kit is optimized for the co-extraction of RNA and DNA, and enables the simultaneous extraction of RNA and DNA from pathogens, including viruses, bacteria, and fungi, from the same sample. The final products are nucleic acids mixture. If only RNA or DNA is required for downstream applications, an appropriate nuclease treatment can be performed after extraction according to the specific experimental requirements. The treatment conditions should be established based on the downstream application and the nuclease manufacturer's instructions and validated experimentally.

What are the possible reasons of low nucleic acid yield when using the NadPure Microbial Pathogens RNA & DNA Kit, and how to solve them?

Samples: Improper sample storage, repeated freeze-thaw cycles, or prolonged storage may compromise nucleic acid stability and reduce yield. It's recommended to store and handle samples according the conditions specified in this user manual, avoid repeated freeze-thaw cycles whenever possible, and prioritize the use of fresh samples. Special sample types should be pretreated as specified. For example, viscous samples (e.g., sputum) should be pre-liquefied according to their viscosity, while whole blood samples should be pretreated by adding an equal volume of 1X PBS or sterile saline. For the other sample types not listed in this user manual, please contact technical support (support@njnad.com) or use a validated pretreatment method. In addition, a low pathogen load in the sample may also result in low or undetectable nucleic acid yield. For these samples, extraction may be performed more than once, and a smaller elution volume may be used to increase the nucleic acid concentration.
Reagents: Precipitates in Patho-Lysis Buffer and Patho-Binding Buffer that have not fully dissolved may affect nucleic acid extraction performance. Inspect these reagents for precipitates prior to use. If present, heat the buffers in a water bath or dry bath at 56°C until the precipitates have completely dissolved. Vortex to mix thoroughly and allow them to equilibrate to room temperature before use.
Operations: 1) Insufficient sample lysis may result in incomplete nucleic acid release. Follow the lysis procedure specified in this user manual, ensuring thorough mixing and compliance with the specified lysis time and operating conditions. 2) Improper handling during washing may result in beads loss. When washing with Patho-Wash Buffer 1 (with absolute ethanol added) and Patho-Wash Buffer 2 (with absolute ethanol added), avoid aspirating or disturbing the beads. Dry the beads for approximately 5 min and avoid over-drying, as this may reduce subsequent nucleic acid recovery.
Temperature: If the actual temperature during lysis and incubation does not reach the set temperature, lysis efficiency may be affected. When using a water bath or dry bath, make sure the actual temperature has reached 56°C as specified in this user manual before incubation, and maintain the specified incubation time.

How effective is the NadPure Microbial Pathogens RNA & DNA Kit for fungi or Gram-positive bacteria with thicker cell walls?

The kit combines mechanical and chemical lysis for sample processing and can be used to extract nucleic acids from micro-organisms with thick cell walls, including fungi (e.g., yeasts and Candida spp.) and Gram-positive bacteria. For these pathogen types, follow the specified lysis procedure to ensure efficient nucleic acid release. Actual extraction performance may vary with sample type, pathogen load, and sample handling etc.

What are the possible reasons of low nucleic acid purity when using the NadPure Microbial Pathogens RNA & DNA Kit, and how to solve them?

What are the possible reasons of downstream application failure when using the NadPure Microbial Pathogens RNA & DNA Kit, and how to solve them?

1) Insufficient nucleic acid yield: Low nucleic acid concentration may not meet the requirements of downstream applications. Check sample quality, pretreatment, lysis, beads binding, and elution. If necessary, interpret the results in conjunction with the internal, positive, or negative control results.

2) Poor nucleic acid purity: Residual wash buffer or other impurities in the elute may interfere with downstream applications. Ensure that wash buffers are prepared correctly, complete all required washing steps, and thoroughly remove residual liquid before elution.

3) Nucleic acid degradation: Improper sample storage, repeated freeze-thaw cycles, or nuclease contamination during the experiment may cause nucleic acid degradation. Store samples and purified nucleic acids as specified in this user manual and avoid repeated freeze-thaw cycles. Use nuclease-free consumables and reagents during the entire workflow, and take precautions to prevent DNase and RNase contamination.

4) Abnormalities or contamination during the workflow: Use appropriate internal, positive, and negative controls according to the specific downstream application to help determine whether the nucleic acid extraction and subsequent experimental procedures have proceeded as expected and to investigate potential contamination.