Cat. No. / ID: 4504065
Cat. No. / ID: 4504263
Cat. No. / ID: 4504063
Cat. No. / ID: 4504163
To ensure highest sensitivity, artus Parvo B19 PCR Kits have been optimized to detect low numbers of parvovirus B19 DNA. The analytical sensitivity of the artus Parvo B19 RG PCR Kit is 0.2 IU/µl in the PCR on the Rotor-Gene 3000.
|Kit||artus Parvo B19 RG PCR Kit|
|Validated sample types||Serum or plasma|
|Analytical sensitivity||0.2 IU/µl in the PCR|
artus Parvo B19 PCR Kits are based on the amplification and simultaneous detection of a specific region of the parvovirus B19 genome using real-time PCR. The kits provide high levels of specificity, sensitivity, and reproducibility.
Each artus Parvo B19 PCR Kit provides 5 quantitation standards (see figure " Reliable quantitation of parvovirus B19 load"). Use of the standards enables accurate quantitation of viral load. In addition, the kits contain a second heterologous amplification system to identify possible PCR inhibition. This is detected as an internal control (IC) in a different fluorescence channel from the analytical PCR. The detection limit of the analytical parvovirus B19 PCR is not reduced.
|Kit||artus Parvo B19 RG PCR Kit|
|Sample types||Serum or plasma|
|Amplicon||76 bp region of the parvovirus B19 genome|
artus Parvo B19 PCR Kits provide all necessary reagents optimized for reliable parvovirus B19 DNA detection and quantitation. Simply add template DNA to the ready-to-use PCR master mix (and Mg solution, LC kit only), and start the reaction on the appropriate real-time cycler using the optimized cycling program described in the kit handbook.For DNA purification, the QIAamp DNA Mini Kit or QIAamp UltraSens Virus Kit is to be used.
|Sample type||Serum, plasma|
|Recommended sample prep||QIAamp UltraSens Virus Kit|
|What detected||Parvovirus DNA|
|Thermal cycler||ABI PRISM 7000, 7700, 7900HT SDS, LightCycler, Rotor-Gene 3000|
• LC: 0.1 - 20.0°C/s
• RG: 2.0°C/s (tube temperature) 5.0°C/s (air temperature)
• TM 7000/7900: 2.3°C/s (with 9600 emulation: 1.0°C/s)
• TM 7700: 1.0°C/s
Yes. Please note that:
• Phenol-based extraction methods should be avoided due to the risk of carrying over phenol to the PCR reaction. Phenol is a strong PCR inhibitor and may lead to false negative results.
• Ethanol – which is a washing buffer component of many extraction systems – should be completely removed prior to the final elution step. When using column-based QIAGEN extraction systems this can readily be achieved by an additional centrifugation after the last washing step (13.000 rpm, 3 min, room temperature). Ethanol is a potent PCR inhibitor and may also lead to false negative results.
• When sample material low in DNA/RNA content is used (e.g. CSF, serum, plasma) QIAGEN strongly recommends to add carrier RNA to the extraction procedure (poly(A) RNA homopolymer, Amersham Biosciences, cat. no.: 27-4110-01). This will increase the overall yield of the DNA/RNA of interest.
Yes, please see our Technical Note 'identification, prevention and elimination of DNA and RNA contaminations'.
The addition of carrier nucleic acids increases the yield of extracted DNA/RNA from analytical samples. Especially when starting from samples low in pathogen or DNA/RNA concentration (e.g. cerebrospinal fluid), we highly recommend to use poly(A)-homopolymers (AmershamBiosciences, Cat. no.: 27-4110-02) as a carrier nucleic acid.
Carrier RNA is not required when sample material containing an excess amount of DNA is used, such as stool, blood cells and others.
Please note that some extraction kits, e.g. the QIAamp Viral RNA Mini Kit, already contain carrier RNA (supplement to the lysis buffer). In these cases, additional poly(A) carrier RNA is not required
The majority of kits provide the IC in a separate, green-colored capped tube for the benefit of the customer. The user can include the IC in the extraction procedure, thereby controlling the efficiency of the DNA/RNA preparation and checking for potential PCR inhibition in the subsequent PCR.
Alternatively, the IC may be used for PCR inhibition control only by adding it to the artus® Master directly. The user manual provides corresponding pipetting schemes for both variants
QIAGEN uses a combination of different "Hot-Start" polymerases depending on the requirements of the kit as the artus® Master composition of each kit is tailored individually. In addition, assay systems for the detection of RNA viruses contain reverse transcriptase enzyme(s).
We strongly recommend to use the IC for every single PCR by either adding it to the extraction procedure or to the artus® Master directly (see user manual for pipetting instructions).
A water/buffer control for the analytical PCR including the IC is sufficient as a negative control. The CT values of the IC are expected to be the same.
artus® kits feature a high specificity and sensitivity in pathogen detection. In principle, samples bearing even low pathogen copy numbers can efficiently be analyzed (down to 10-50 copies/PCR).
The IC has carefully been adjusted to ensure that it does not influence the analytical PCR.
A failure of the IC amplification in the absence of an analytical PCR signal is indicative for either a PCR inhibition or a significant DNA/RNA loss during the nucleic acid isolation procedure (provided the IC was added to the extraction).
Various reasons could account for a failure of IC amplification.
• Inhibition of PCR. PCR inhibitors were not quantitatively removed during DNA/RNA extraction. This may particularly occur with stool or urine as sample materials which contain a large number of partially unknown potential PCR inhibitors. Transfer of such molecules may impair or eliminate the DNA polymerase activity. Alternatively, the incomplete removal of ethanol (as a component of washing buffers of many extraction protocols such as the QIAGEN column-based extraction systems) may have caused PCR inhibition. If QIAGEN extraction systems are used, this can be prevented by performing an additional centrifugation step prior to the final elution step (please refer to the artus® user manuals for further details).
• Loss of IC during extraction. The use of a non-recommended extraction kit or non-compliance with the extraction kit protocol may lead to loss of DNA/RNA and, consequently, of the IC – provided the IC was added to the extraction procedure. This risk of DNA/RNA loss can significantly be decreased by adding carrier RNA to the extraction. If carrier RNA is not supplied with the extraction kit, QIAGEN recommends the use of homopolymer poly(A) RNA (Amersham Biosciences, cat. no.: 27-4110-01).
• Addition of IC to extraction prior to initial lysis step. It is important that the IC is added only after the sample material has been combined with the lysis buffer of the extraction system. If the IC is added prior to this lysis step it may be digested by DNases/RNases present in the sample material. The lysis buffer contains protein-denaturing agents which inactivate all DNases/RNases promptly.
Manufacturing failure/faults or air bubbles in the tubes and/or capillaries may cause initial fluorescent signals that are too high. When using artus® kits designed for the Rotor-Gene instrument with a total volume of 50 μl, the 20 μl sample has to be mixed with 30 μl of artus® Master Mix in the reaction tube by pipetting up and down several times. Otherwise, distribution of fluorescence is not homogeneous within the first few PCR cycles which may lead to initial fluorescence signals that are too high.
Yes. The capillaries have to be carefully opened, put upside down in a conventional 1.5 ml reaction tube and centrifuged briefly at 2.000 rpm.
Please note that a gel electrophoresis analysis should be performed in a separate room to avoid contamination by the amplicon
All artus® PCR kits for RNA-based detection of pathogens contain heterologous in vitro transcribed RNA as an internal control whereas DNA PCR kits contain a corresponding heterologous DNA internal control. The concentration of the IC template as well as the length of the IC PCR product may vary from kit to kit.
Air bubbles in the reaction solution may cause a strong fluorescence decrease at the beginning. In the Rotor-Gene® instrument those bubbles will be removed by the spinning action of the instrument. Insufficient mixing of sample and Rotor-Gene® artus® Master Mix may also lead to a steep initial fluorescence decrease due to an uneven distribution of the fluorescent dyes inside the tube.
As mentioned in the user manual, QIAGEN strongly recommends to thoroughly mix those two fractions (by pipetting up and down several times) prior to the PCR run.
The precision of the artus® kits is determined by measuring the
These three variabilities are stated as coefficients of variation (in percent) and allow the determination of the total variance which is indicative for the reliability of the detection system.
In principle, adjustment of DNA concentration prior to the PCR setup is not required. The linear quantitation range of artus® kit systems comprises four to five logarithmic orders of magnitude, as defined by the quantitative standards (QS) series.
Most diagnostic samples lie within this linear range. In HBV or Parvo B19 diagnostics positive material with unusually high viral loads may be observed which result in CT values lower than the highest QS (e.g. QS1 for HBV: 105 IU/μl), indicating that those samples have a virus titer that exceeds the first QS.
Since precise quantitation is not possible outside the linear range of the standard curve, a repetition of the PCR using diluted sample DNA is recommended.
In general, the following pipetting order is applicable:
To avoid cross-contamination deriving from the supplied QS, QIAGEN strongly recommends to pipette and seal (if possible) the analytical patient samples (2.) prior to the QS (3.).
A water control should be included subsequently to pipetting the analytical/QS samples (4.) to control a contamination-free process flow.
Since May 2002 artus has produced and inspected its products in accordance with the European guidelines (directives, standards) for in vitro diagnostics (directive 98/79/EG IVD).
Thus, they are allowed to carry the CE-label (directive 93/42/EWG MPG). A prerequisite for this is the maintenance of a certified quality management system according to DIN EN ISO 9001 as well as a running quality and conformation control for each lot produced.
The specificity of the detection system for a particular pathogen is ensured by the careful selection of highly specific primers and probes as well as by stringent reaction conditions. The sequence specificity of the primers and probes is verified by homology searches and sequence alignments with the pathogen's and closely related sequences.
The specificity is further controlled by assaying the kit system with isolates of closely related pathogens, pathogens causing similar symptoms, and, if applicable, with isolates of different genotypes/subtypes of the pathogen of interest.
For a variety of pathogens (HIV, HBV, HCV, Parvovirus B19, HAV) international units (IU) have been determined by the World Health Organisation (WHO) for the establishment of worldwide comparable pathogen diagnostics. Conversion factors are documented in a WHO report to allow a recalculation from IU/vol. to copies/vol. ("WHO Consultation on International Standards for in vitro Clinical Diagnostic Procedures based on Nucleic Acid Amplification Techniques (NAT)", 2002). However, QIAGEN considers the conversion of IU into copies not permissible or, at least, misleading. The conversion of concentration units is thought to allow a comparison of results generated with different detection assays. However, since the quantitation standards of many competitive products are not calibrated with international standards, these conversions may lead to discrepant results. Hence, these conversions do not contribute to a comparability of different assay results but may rather obscure conclusions. QIAGEN calibrates all artus® quantitation standards using international standards if available (e.g. from the National Institute for Biological Standards and Control, UK; www.nibsc.ac.uk). If the quantitation standards of a competitive product were adjusted correspondingly, the assay results may indeed be compared. Please also note paragraph 3.2.2. in the European "IVD guideline on common technical specifications for in vitro-diagnostic medical devices" which clearly states that international reference material has to be used for detection limit determination:
"The analytical sensitivity or detection limit for NAT assays shall be expressed by the 95 % positive cut-off value. This is the analyte concentration where 95 % of test runs give positive results following serial dilutions of an international reference material for example a WHO standard or calibrated reference materials."
For increased stability of the kit components the Mg2+ solution has been taken out of the artus® Master solution.
In general, all run files and result data should be forwarded.
For each real-time instrument these are the following files:
ABI PRISM®: *.SDS files
Rotor-Gene®: *.REX or *.REA (Rotor-Gene® archive) files
LightCycler® 1.x: *.ABT and *.FLO and *.TEM
LightCycler® 2.0: *.IXO files (ccc-file has to be imported into the file)
Generally, we recommend to forward all run files in a compressed file format (*.zip files) using the Winzip software (free download available at www.winzip.com). This prevents the files (especially LightCycler® files) from being automatically unpacked by e-mail browsers.
For the analysis of Rotor-Gene® files we recommend to save the experiment and forward the data as a compressed experimental archive file (*.REA).
Conventional DNA polymerases exert a certain level of background activity at room temperature. This may affect the performance of the individual PCR reactions.
During setup some minor amplification may occur prior to starting the PCR instrument. To prevent any background polymerase activity and to synchronize all reactions, the enzyme is silenced either by binding of a specific antibody or by chemical modification.
This enzyme inhibition can be released by an initial denaturation step at 95°C.
With a few exceptions all artus® kits have been equipped with 4-5 quantitation standards (QS 1-4/5) depending on the individual kit's requirements. These QS tubes (red colored caps) contain plasmid DNA of defined concentrations (given in either copies/μl or IU/μl) in which the pathogen-specific PCR product was cloned.
For artus® RNA virus detection kits (West Nile Virus, Enteroviruses, HAV etc.) in vitro transcribed RNA obtained from cloned plasmids is provided. The plasmid concentrations have been determined by fluorimetry or calibrated with international standards where possible (e.g. HBV). This QS series allows plotting a standard curve for the determination of precise pathogen loads (stated in copy or IU values).
• Stick to a three room strategy: Laboratory 1: nucleic acid isolation
Laboratory 2: PCR setup
Laboratory 3: PCR instrumentation/PCR run
-> if three rooms are not available, please perform nucleic acid extraction in
a facility spatially separated to the PCR laboratory
• Use pipette tips with aerosol barriers only.
• Store positive controls separately from negative controls.
• Always pipette positive after the unknown samples.
• After each pipetting of an unknown sample seal the reaction tube/capillary.
• Do not reuse capillaries, tubes, filter tips etc.
• Use different sets of pipettes for aliquotting artus® kit components and handling controls.
• In case of capillary breakage or when tubes unseal, clean the work area, including the thermocycler, with sufficient amounts of a DNA-decontaminating agent (hypochlorite or e.g. DNA-Zap by Ambion) or UV light.
• The artus® Master was not completely thawed and as a result the concentration is incorrect.
• The artus® Master was not mixed sufficiently by vortexing.
• The Mg2+ solution was not completely mixed/thawed (where applicable) before being added to the artus® Master.
• The solutions were not sufficiently mixed and then centrifuged briefly.
• The PCR setup was not carried out on ice or in a cooler.
• The solutions were frozen and thawed too often (if possible do not thaw more than twice).
• During transportation the kits were not kept frozen permanently.
• Pipettes are not calibrated correctly.
• Pipette tips do not fit the pipettes.
• An extraction procedure other than those recommended was used.
• The extraction volume has been increased.
The IC is a heterologous amplification system with a unique set of primers and probes. Other competitive products frequently utilize a homologous IC system with identical primer binding sites which may lead to primer binding competition and may, in consequence, negatively affect the detection sensitivity.
With artus® kits such primer binding competition can be excluded since the amplified sequences differ.
Storage must be performed at -20°C, transport is possible at -80°C (dry ice). Repeated freeze-thaw cycles (>2x) of kit components should be avoided as this may negatively affect the kit sensitivity. If the kit is used only intermittently, we recommend to aliquot all reagents (artus® Master, Internal Control, Quantitation Standards) in a desired number of tubes. This aliquoting should be carried out for all kit reagents upon receipt of the kit.
Interpretation of the IC results strongly depends on the analytical PCR data. High pathogen copy numbers in a sample inevitably lead to impairment or, in rare cases, to even a complete failure of the IC PCR (as a consequence of competition between the two PCR systems).
The impairment is reflected in a decrease of the IC fluorescence intensity. However, the CT value is not affected. In contrast, PCR inhibition can lead to an increase of the CT value, i.e. to a significantly less efficient PCR. Since PCR inhibition can lead to false negative results, it is advisable to repeat the particular sample run and/or modify the RNA/DNA extraction procedure. However, the curve shape and CT values of the other ICs of the same run should be taken into account for comparison. If only one IC PCR differs from the others, an inhibition is very likely. If the IC was included in the extraction procedure of the sample material, a CT deviation of the IC may also indicate a partial IC loss and, hence, a deficient DNA/RNA preparation. In this case, the nucleic acid preparation should be repeated
The shelf-life of each artus® kit is stated on the kit box label and can range from three to thirteen months at -20°C after production date.
Since QIAGEN usually produces small lots, the shelf-life after kit delivery is not reduced significantly.