artus WNV LC RT-PCR Kit CE

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

As part of the internal validation the analytical sensitivity is determined by a probit analysis. This is performed by setting up six to eight dilution series (usually in half-logarithmic concentration steps) of the plasmid cloned PCR product starting with the lowest concentration of quantitation standard. Each of these dilutions is run on three different days in eight-fold replicates. From these data the detection limit is calculated using a statistical algorithm; the probit analysis.

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 recommends to use collection tubes with EDTA or sodium citrate additives as anticoagulants. In contrast, heparin blood is not suited for artus PCR analysis as this anticoagulant is known as a potent PCR inhibitor.

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).

 

Yes. However, for melting curve analysis the calculation method "Linear" has to be selected. Otherwise melting curves are calculated with the "Polynomial" method.

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.

Since artus^® kits allow an extraction efficiency control by adding the IC (provided in a separate tube) to the extraction procedure and are optimized for quantitation using the QS directly for the real-time PCR, the QS must not be included into the extraction process

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.

No. For quantitation of ABI PRISM^® PCR results, the artus^® quantitation standard series has to be included in parallel in each real-time PCR run.

In contrast to the 7900HT and the 7700 instruments which use argon lasers as a light source, the ABI PRISM® 7000 SDS instrument works with a Tungsten halogen lamp for excitation of the fluorescent dyes. Detection of individual fluorescent signals is mediated by a four filter system.

The precision of the artus^® kits is determined by measuring the

• intra-assay variability: deviation of Ct values of a standard of defined concentration (in eight-fold replicates) within one run
• inter-assay variability: deviation of Ct values of a standard of defined concentration on three instruments, performed by three different lab workers
• inter-lot variability: deviation of Ct values of a standard of defined concentration in three PCR runs using three different batches of as many kit components as possible; obligatorily, three different primers and probes batches have to be used for the determination of the inter-lot variability

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: 

1.  artus® Master Mix 
2.  analytical samples
3.  quantitation standards (QS, supplied) 
4.  nuclease-free sterile water (supplied)

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."

A high CT value indicates a positive sample of correspondingly low concentration. On the basis of the CT values of an external standard, the real-time PCR instrument software calculates the amount of the sample's input DNA/RNA. However, if a CT value of a particular sample is higher than that of the last quantitation standard (QS4 or QS5) a precise quantitation of this sample may not be possible. Nevertheless, it still has to be reported as positive.

The 7700 instrument employs a different smoothening algorithm of the curves, which results in more evenly shaped amplification curves.

Yes. The fluorescence in each well of a 96 well plate is measured individually by glass fiber optics. Therefore, damage or impairment of one of the fibers may lead to deviating results.

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.

No. The brief centrifugation steps do not impair the performance of the probes.

• 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.

CT is the abbreviation for Threshold Cycle and is defined as the calculated cycle number at which the PCR product crosses a threshold of detection. This threshold line is either automatically set by the software algorithm of the real-time PCR instrument or can be manually adjusted. The CT values are essential for quantitation since the standard curve is generated by plotting the CT values versus the logarithmic concentration. A positive unknown sample will also be assigned a specific CT value and by comparison with the standard curve a particular concentration can be calculated

Although a selftest can be run with an inserted carousel, we strictly recommend to run the selftest without the carousel filled with analytical samples. During the selftest the LightCycler^® instrument is checked for optimal heating and cooling performance so that anything inside the thermal chamber will be subject to a brief temperature variation. In theory, these short heating/cooling steps could have an influence on the artus^® kit performance.

Yes. The sample sheet can be amended during or after the run by activating the "samples" software button. The sample sheet will open and may be modified.

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.