For isolation of viral RNA and DNA and bacterial DNA from a variety of animal sample types
Effective April 1, 2018, product available exclusively from INDICAL BIOSCIENCE (formerly QIAGEN Leipzig). Product has been rebranded to “IndiSpin Pathogen Kit”. Learn more.
The QIAamp cador Pathogen Mini Kit simplifies the isolation of viral RNA and DNA and bacterial DNA from a whole range of animal samples, including undiluted whole blood, serum, swabs, and tissue. Using a single, rapid spin-column procedure, contaminants and inhibitors are removed to yield pathogen nucleic acids that are ready for use in downstream applications, such as real-time PCR and RT-PCR. The procedure can be fully automated on the QIAcube.
Data show that high and identical purification efficiency can be achieved with both the manual and automated protocols for viral RNA isolation (see the figure "Comparison of manual and automated processing."). Bovine serum was spiked with virus particles from cell culture. The sample was serially diluted 1:10 with bovine serum and processed in duplicate using the QIAamp cador Pathogen Mini Kit either manually or using the automated protocol on the QIAcube. Viral RNA was detected using the QuantiFast Pathogen RT-PCR +IC Kit with target-specific primers and probe.
The robustness of the protocol is demonstrated by the high intra- and inter-assay repeatability (see the figure "High repeatability of nucleic acid isolation."). Whole blood samples from different animals were spiked with virus particles from cell culture and then frozen at -20°C. Three replicates for each sample were processed on three different days with the QIAamp cador Pathogen Mini Kit. The viral RNA was amplified with the QuantiFast Pathogen RT-PCR +IC Kit using target-specific primers and probe.
The kit also successfully isolates bacterial DNA from animal samples, as shown in the figure "Successful isolation of bacterial DNA". Different animal samples were spiked with Gram-negative or Gram-positive bacteria and pretreated as necessary (see the table "Pretreatments for the QIAamp cador Pathogen Mini Kit protocol"), and then the automated isolation protocol was run on the QIAcube. The bacterial DNA was successfully purified. PCR setup with parallel preparation of reaction mixes for both bacterial targets was performed using the QIAgility, and the bacterial DNA was amplified with the QuantiFast Pathogen +IC Kit with target-specific primers and probes.
The QIAamp cador Pathogen Mini Kit combines the features of several sample preparation kits based on spin-column technology to permit the purification of viral RNA and DNA and bacterial DNA from animal fluid and tissue samples. The spin-column procedure uses optimized buffers and enzymes to lyse samples and stabilize the pathogen nucleic acids. This RNA and/or DNA binds to the QIAamp silica membrane, while contaminants pass through. Wash buffers are used to completely remove PCR inhibitors, such as divalent cations and proteins, and the pure pathogen nucleic acids are then eluted in Buffer AVE.
QIAamp cador Pathogen Kit technology yields pathogen RNA and DNA from animal samples that is ready for use in downstream applications such as real-time PCR and RT-PCR.
In contrast to other kits based on silica membranes, up to 200 µl of undiluted animal whole blood samples can be processed with the QIAamp cador Pathogen Mini Kit without clogging the filter. See the table "Key features of the QIAamp cador Pathogen Mini Kit".
| Format and processing | Mini spin columns, manual centrifuge protocol, or automated centrifuge protocol using the QIAcube |
| Target nucleic acids | Viral RNA, viral DNA, and bacterial DNA |
| Sample source types | Animal whole blood, serum, plasma, other fluids, swabs, washes, and tissues |
| Sample size | Up to 200 μl or 25 mg |
| Preparation time | 20 to 40 minutes |
| Elution volume | 50 to 150 μl |
Most common fluid samples can directly be processed using the main protocol, which involves the lysis, binding, wash, and elution steps described in the "Principle" section.
Tissue samples and samples containing difficult-to-lyse bacteria require the appropriate pretreatment (see the table "Pretreatments for the QIAamp cador Pathogen Mini Kit protocol"). After pretreatment, the samples undergo the same procedure as for fluid samples (see the flowchart "Processing of various sample types and targets"), enabling parallel processing either manually or in an automated procedure on the QIAcube.
| Name | Application |
|---|---|
| Pretreatment B1 | For difficult-to-lyse bacteria* in whole blood or pretreated tissue |
| Pretreatment B1 | For difficult-to-lyse bacteria* in whole blood or pretreated tissue |
| Pretreatment B2 | For difficult-to-lyse bacteria* in cell-free fluids |
| Pretreatment B3 | For easy-to-lyse bacteria in high-volume cell-free fluids |
| Pretreatment T1 | Mechanical disruption of tissue for extraction of viral RNA and DNA |
| Pretreatment T2 | Enzymatic digestion of tissue for extraction of viral and bacterial DNA |
| Pretreatment T3 | Rapid partial disruption of tissue for extraction of viral RNA and DNA and bacterial DNA |
| Pretreatment T4 | Organic extraction for extraction of viral RNA and DNA and DNA from easy-to-lyse bacteria* from difficult tissue |
The QIAamp cador Pathogen Mini Kit yields pure, high-quality viral RNA, viral DNA, and bacterial DNA that is suitable for a wide range of downstream applications, including real-time PCR and RT-PCR for:
Whole blood samples from various animals were spiked with RNA virus particles. The samples were processed in triplicate on 3 separate days. The data from the RT-PCR show high repeatability with error bars representing ±1 S.D.
