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 HPA-Coronavirus Real-time RT-PCR Reagents

Quantitative in vitro Diagnostic kit for use with a Real-time RT-PCR Cycler  (for research use only)

Cat. Nr. :  CorQPCR-25

Price : 875 Euro / kit

1. Contents

Preparations 25

HPA-Coronavirus Master 27 µl

MgCl2(25 mM) 108 µl

P+P Mix 270 l HPA-Coronavirus QC 1 (101 copies/ l)* 150 µl HPA-Coronavirus QC 2 (102 copies/ l)* 150 µl

HPA-Coronavirus QC 3 (103 copies/ l)* 150 µl

HPA-Coronavirus QC 4 (104 copies/ l)* 150 µl

HPA-Coronavirus QC 5 (105 copies/ l)# 150 µl

RNase free dH2O 1000 µl

*: pBluesript SK with an insert of specific SARS cDNA fragment. Used as positive controls,

#: SARS RNA in vitro transcribed from pBluescript SK with an insert of specific SARS cDNA fragment, 105 copies / l of SARS RNA transcripts. Used for checking the efficiency of trasnscriptase.

2. Storage

The kit components should be stored at -20? and are stable for 3 months at this temperature. Repeated thawing and freezing (> 2 ×) should be avoided, as this may reduce the sensitivity. If the kit is to be used only intermittently, the reagents should be frozen in aliquots. Storage at +4? should not exceed a period of 5~6 hours.

3. Additionally Required Materials and Devices

(1) Disposable powder-free gloves

(2) Coronavirus RNA Purification Kit (see 8.1 RNA Purification)

(3) Physiological salt solution (0.9 % NaCl) containing 1 % N-Acetylcystein

(4) Pipettes (adjustable 1~20 µl)

(5) Sterile pipette tips with aerosol barrier

(6) Vortex mixer

(7) Desktop centrifuge with rotor for 2 ml reaction tubes (

8) Real-time RT-PCR Instrument

4. General Precautions for PCR

The user should always pay attention to the following:

(1) Use pipette tips with filters.

(2) Storage of positive material (samples, controls and amplicons) should be separated from all other reagents and they should be added to the reaction mixes in a spatially separated facility.

(3) Thaw all components thoroughly at room temperature before starting an assay.

(4) When thawed, mix the components and centrifuge briefly.

(5) Work quickly on ice or in the Cooling Block.

5. Pathogen Information

Coronaviruses, a genus in the family Coronaviridae, are large enveloped, positive-stranded RNA viruses that cause highly virulent disease in humans and domestic animals. Two Coronaviruses that are known to infect humans cause one third of common colds and are also a common cause of health care-associated upper respiratory infections in premature infants. A member of the Coronavirus family is considered to cause the Severe Acute Respiratory Syndrome (SARS). The virus is not classified yet. In the literature its suggested name is Human Pneumonia-Associated Coronavirus (HPAC). A part of a putative Coronavirus polymerase gene was identified via PCR in a SARS patient by the Bernhard Nocht Institute for Tropical Medicine in Hamburg and cooperating laboratories. This assay was used to establish a commercially available real time RT-PCR system for the direct detection of this new Coronavirus species.

Attention: Cross reactivity of this amplification system has not been fully determined and one has to bear in mind a possible amplification of other species of the genus Coronaviridae.

In addition: The WHO case definition does not include a positive PCR-result for the diagnosis of SARS.

6. Principle of Real-Time PCR

Pathogen diagnosis by the polymerase chain reaction (PCR) is based on the amplification of specific regions of the pathogen genome. In real-time PCR, the amplified product is detected via fluorescent dyes. These are usually linked to oligonucleotide probes which bind specifically to the amplified product. Monitoring the fluorescence intensities during the PCR run (i.e. in real-time) allows the detection and quantification of the accumulating product without having to re-open the reaction tube after the PCR run.

7. Product Description

The HPA-Coronavirus Real-time RT-PCR Reagents constitutes a ready-to-use system for the detection of HPA-Coronavirus RNA using PCR in the Real-time RT-PCR Instrument. The HPA-Coronavirus Master contains reagents and enzymes for the specific amplification of a 80 bp region of the HPA-Coronavirus genome. Positive controls (HPA-Coronavirus QC 1-5) are supplied which allow the determination of the pathogen load. For further information, please refer to section 8.2 Positive controls.

8. Protocol

8.1 RNA Purification Gentaur offers a Kit for Rapid Enrichment of SARS Virus, which can enrich and partially purify SARS virus, and Coronavirus RNA Purification Kit, which can obtain viral RNA free from contamination with protein, pigment, lipid and RT-PCR inhibitors. The concentration of HPA-Coronavirus is very low in most of clinical samples. It is recommended to enrich virus with Kit for Rapid Enrichment of SARS Virus before purification of viral RNA.

8.2 Positive controls Positive controls (HPA-Coronavirus QC 1-5) are supplied with gradient concentration. HPA-Coronavirus QC 1-4 contain a specific region of the HAP-Coronavirus genome cloned in pBS plasmid. HPA-Coronavirus QC 5 is RNA templates transcribed from pBS plasmid and used for positive control and checking the efficiency of trascriptase.

8.3 Quantification The enclosed quantification standards (HPA-Coronavirus QC 1-5) are amplified as previously purified samples and the same volume is used. To generate a standard curve in Real-time RT-PCR Instrument, all 5 quantification controls should be used and defined in the Sample Loading Screen as standards with the specified concentrations. The standard curve generated as above can also be used for subsequent runs, provided that at least one standard is used in the current run. For this purpose, the previously generated standard curve needs to be imported. However, this quantification method may lead to deviations in the results due to variabilities between different PCR runs. Attention: The quantification controls are defined as copies/ l. The following formula has to be applied to convert the values determined using the standard curve into copies/ml of sample: Result (copies/ml) = [Result (copies/ l)×Elution Volume (ml) ]/Sample Volume (ml)

8.4 Preparing the PCR Please make sure that at least one quantification standard as well as one negative control (water, PCR grade) are included per PCR run. To generate a standard curve, use all supplied quantification standards (HPA-Coronavirus QC 1-5) for each PCR run. Before each use, all reagents need to be thawed completely and mixed (by pipetting repeatedly up and down or by brief vortexing).

1. Preparation of Master Mix HPA-Coronavirus Master MgCl2(25 mM) P+P Mix

1 µl 4 µl 10 µl

2. Preparation of PCR assay Master Mix RNA sample (or HPA-Coronavirus QC1-5, dH2O ) Total volume 15 µl 5 µl 20 µl

Close the PCR tube, add mineral oil and short spin in a desktop centrifuge.

8.5 Programming of the Real-time RT-PCR Instrument

A. cDNA synthesis and pre-denaturation: 45?, 15 min 94?, 3 min

B. PCR amplification Perform 35 cycle of: 94?, 10 sec 58?, 30 sec

C. Fluorescence measure Perform at 58?.

9. Specificity

The primers and probes are checked for possible homologies to other known sequences by means of sequence comparison. The detectability of all relevant subtypes and genotypes has thus been ensured.

10. Product Use Limitations

All reagents may be used exclusively in in vitro diagnostics for research only. (1) The product is to be used by personnel specially instructed and trained in the in vitro diagnostics procedures only. (2) Compliance with the kit protocol is required. (3) Attention should be paid to expiration date printed on the kit box. Do not use expired components.

 

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Last modified: 05/19/16