Background:
Respiratory syncytial virus (RSV) causes infections of the lungs and respiratory tract. It’s so common that most children have been infected with the virus by age of two. Respiratory syncytial virus can also infect adults. In adults and older, healthy children, RSV symptoms are mild and typically mimic the common cold. Self-care measures are usually all that’s needed to relieve any discomfort. RSV can cause severe infection in some people, including babies 12 months and younger (infants), especially premature infants, older adults, people with heart and lung disease, or anyone with a weak immune system.
Test Principle:
The DiAGSure® Respiratory Syncytial Virus (RSV) Detection Assay (Multiplex, TaqMan based) is an in vitro diagnostic (IVD) real-time polymerase chain reaction (qPCR) test, involving TaqMan chemistry intended for the qualitative detection of RNA specifically from Respiratory Syncytial Virus collected from blood specimens from individuals with signs and symptoms of infection of the disease. On the other hand, this test is significantly important for those who are suspected of flu, common cold and bronchitis related diseases even pneumonia by their health care provider. This Multiplex PCR includes two targeted gene specific to respiratory syncytial virus and one internal control in a single-tube reaction.
Sample types:
Blood specimens
Estimated operating time:
~1.4 hrs.
Cap | Contents | Description | 20 Tests | 50 Tests | 100 Tests |
GDQ2010-20R | GDQ2010-50R | GDQ2010-100R | |||
Amber | 2X one-step Master Mix | Amplification Mix | 300 µL | 700 µL | 1.4 mL |
Amber | Primer-Probe Mix | Amplification Reagent | 50 µL | 120 µL | 240 µL |
Yellow | Positive Control (PC) | Positive Control | 100 µL | 200 µL | 400 µL |
Blue | Negative Control (NC) | Nuclease Free Water | 1 mL | 1 mL | 1 mL |
White | RNA Dilution Buffer | Tris-based buffer | 200 µL | 500 µL | 1 mL |
Materials required but not provided
Reagents
• DNA extraction components: GSure® Viral DNA Isolation kit (from plasma/serum/body fluid).
• DNase free/sterile PCR grade water
Consumables
• Disposable tips containing hydrophobic filters
• Sterile DNase-free 1.5 ml vials (Eppendorf tubes)
• Suitable DNase-free 0.1 or 0.2 ml PCR tubes for use of RGQ instruments
• Suitable multiwell 96 plates (Roche, product code 04 729 692 001) for use of LC480 II instruments
• DNase-free Mic tubes and caps (BMS, ref 60653) for use on the Mic qPCR cycler
• Hard-shell® PCR plates 96-well, thin wall (Bio-Rad, Hsp9655) for use on the CFX96TM
• MicroAmp Fast Optical 96-well reaction plate 0.1 ml (Applied Biosystems, ref 4346906) for use on the QS5
Equipments
• Adjustable pipettes: 0.1-2 μl, 2-20 μl, 20-200 μl, 200-1000 μl
• Tube rack for 1.5 ml vials
• Cooling block (1.5 ml) or ice (for placing kit components during reaction set up)
• PCR cooling block or ice for PCR reaction tubes and 96-well plates
• Vortex mixer
• Benchtop centrifuge with a rotor for 1.5 ml tubes
• Centrifuge suitable for PCR plates
• Calibrated real-time PCR instrument
• Automated DNA extraction system
Kit Stability:
• Kit is shipped on dry ice and should be stored immediately upon receipt at –20°C in a constant temperature freezer. Please refer to product label for final expiry date.
• This product can be used for 30 days after opening the vials.
• This product can be used for maximum 7 repeats of freezing and thawing.
Specimen handling & Storage:
After sample collection, perform the test on the same day. Otherwise, store it in the following condition: 2 to 8˚C for no more than 24 hours. It should be stored below -20 ˚C for not more than 3 days, and can also be stored for a long time below -70 ˚C. Repeated freezing-thawing should be avoided.
Transportation:
The foam box is sealed with ice for transportation.
A. Sample solution preparation:
Clinical samples are extracted according to the corresponding requirements and steps with the RNA extraction kit, and the extracted RNA could be directly used for detection. If samples are not immediately tested after extraction they can also be stored at -70°C and repeated freezing and thawing should be avoided. It is advised to start amplification with at least 100 ng of RNA.
Note: Freshly extracted RNA should be used. Otherwise, RNA should be stored in alcohol at -80°C. Since RNA is a labile biomolecule, improper handling and storage can lead to significant loss of titer and may affect the kit’s sensitivity.
B. Preparation of amplification reagent:
Take the reagents out from the -20˚C freezer and thaw them on ice. After thawing, briefly vortex the reagents followed by a short spin. The RT and PCR mixes should be kept on ice.
C. qPCR protocol:
Per sample, set up a single reaction according to the following table.
Components | Volume per reaction |
2X One-step Master Mix | 13 µL |
Primer-Probe Mix | 2 µL |
Extracted RNA/ Positive Control (PC)/Negative Control (NC) | 10 µL |
Total Volume | 25 µL |
D. Real-time PCR Instrument set up:
Set up the PCR tubes/strips/plate in the real time machine and label the sample slots accordingly. Select the sample type (Unknown/PC/NTC) and select the acquisition channel for each slot as follows:
Gene | Channel | Source wavelength (nm) | Detection wavelength (nm) |
Gene-1 | FAM (Green) | 470 | 510 |
Gene-2 | HEX (Yellow) | 530 | 555 |
IC | Cy5 (Red) | 651 | 670 |
E. PCR cycling conditions:
Stage | Step | Temperature (°C) | Time | No. of cycles |
Hold stage | 50 | 10 min | 1 | |
Hold stage | 95 | 3 min | 1 | |
PCR Stage | Step 1 | 95 | 10 sec | 40 |
Step 2 | 60* | 30 sec |
*Acquisition step (Green/Yellow/Red Channel)
F. Instrument compatibility
The DiAGSure® Respiratory Syncytial Virus with a broad range of real-time PCR platforms. Performance of the kit has been verified on the following systems:
Applied Biosystems® 7500/7500 Fast Real-Time PCR System, Quanstudio 3, 5, Bio-Rad CFX96, Qiagen Rotor Gene Q, Roche Lighcycler® 480.
G. Performance Evaluation:
I• Specificity:
The target sequences detected in this kit are the conserved regions of RSV-A and RSV-B. These primer sets used in the assay failed to give any amplification from human genomic RNA extracted from blood eliminating the possibility of non-specific annealing with human RNA. Furthermore, the primers did not cross-react with other pathogens with similar symptoms.
II• Detection limit:
The absolute sensitivities were obtained using synthetic RNA as a template for detection. The Limit of Detection (LoD) was found to be 10 copies of RNA per reaction for both genes under in vitro conditions.
III• Safety Information:
The DiAGSure® Respiratory Syncytial Virus Detection Assay (Multiplex, TaqMan based) is for laboratory use only. Use proper safety measures while handling clinical samples, like wearing mask, gloves, lab-coat, etc.
IV• Ct cut-off for each fluorescent channel:
Target | Ct Value | Interpretation |
Gene1 (FAM) | Ct≤36 | RSV-A Gene Positive |
Gene2 (HEX) | Ct≤36 | RSV-B Gene Positive |
IC (Cy5) | Ct≤35 | Internal Control Positive |
V• Interpretation of the results:
Assessment of clinical specimen test results should be performed after the positive and negative controls have been examined and determined to be valid. If the controls are not valid, the patient results cannot be interpreted.
Refer to the table below for the validity and the interpretation of each specimen result according to the results of each channel.
Target | Gene-1 | Gene-2 | IC | Interpretation | Results |
Fluorophore | FAM/Green | HEX/Yellow | Cy5/Red | ||
Case-1 | Positive | Negative | Positive | RSV-A Positive | All Target Results are valid. Result for RSV-A RNA is Detected. |
Case-2 | Negative | Positive | Positive | RSV-B Positive | All Target Results are valid. Result for RSV-B RNA is Detected. |
Case-3 | Negative | Negative | Positive | RSV Negative | Result for RSV RNA is Negative or below detection limit. |
Case-4 | Negative | Negative | Negative | Invalid | Sample should be repeated once from extraction. If a second failure occurs, it is reported to sender as invalid and recommend recollection if patient is still clinically indicated. |
* If the target gene signal (HEX Channel) is strong, the IC (Cy5 Channel) may be negative.
Performing lab activities should always be done in compliance with general safety regulations. For more information on chemicals, consult the appropriate material safety data sheets (MSDS), which is part of the kit insert.
The following precautions should be taken to both avoid contamination and allow optimal performance and reproducibility of the assays:
• The PCR assay should only be performed by qualified laboratory personnel.
• When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.
• Physically separate the workplaces as outlined in table 3.
• Use disposable tips containing hydrophobic filters to prevent cross-contamination.
• Use DNase-free PCR vials.
• Thaw DNA samples always on ice and keep them on ice or on a cooling block.
• Keep enzymes always on ice or on a cooling block when taken out of the freezer. Handle enzymes with care and mix very gently.
• When thawed, spin down the reagents for 5 seconds in a centrifuge and mix by gently pipetting up and down.
• The cycling program should be programmed in the real-time PCR instrument before performing the assay.
• Spin down the PCR mixtures shortly in the PCR plate before transferring to the real- time cycler (not necessary for RGQ).
• Do not open the PCR vials/plates after PCR amplification.
Reagent storage and handling
Table 3. Handling procedures in different areas
Location | Handling |
Area 1 | WRTaqMan Master Mix, Primer-probe mix |
Preparation of PCR mix | |
Aliquoting of PCR mix in respective wells | |
Addition of Negative Control (NC) | |
Area 2 | Storage of Quantitative Standards |
DNA extraction from samples | |
Adding DNA-extracts to the real-time PCR mix | |
Adding Standards to the real-time PCR mix | |
Area 3 | Real-time PCR reaction |
It is recommended to perform all activities in laminar air flow safety cabinet II in order to minimize the chance of cross-contamination.
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Efficient and reliable PCR purification kit ensuring high-yield, high-purity DNA for accurate downstream applications.
High-yield, high-purity RNA isolation from blood samples for accurate and reliable gene expression and molecular analysis.
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