Background:
Rhinovirus is a virus type of the Picornavirus family that causes many common colds and upper respiratory infections. Less commonly, these viruses may cause bronchiolitis or pneumonia. Rhinoviruses are single-stranded RNA viruses and over 100 types identified. Rhinovirus infection is easily spread from person-to-person. Symptoms of rhinovirus infection usually develop within 12-72 hours of exposure.
Test Principle:
The DiAGSure® Rhinovirus detection PCR assay can detect Rhinovirus and is a much more sensitive and fast detection method for the detection of viral infection. The DiAGSure® Rhinovirus Infection Detection Kit (Multiplex, TaqMan based) is an in vitro diagnostic (IVD) Real-time Reverse Transcriptase-polymerase chain reaction (qRT-PCR) test involving TaqMan chemistry intended for the detection of viral RNA in sputum samples. This two-plex PCR detects the 5’ Untranslated region (UTR) of the viral genome and includes an internal control (IC) reference gene in a single-tube reaction.
Sample types:
Blood plasma/sputum/stool/oro-pharyngeal swab samples
Estimated operating time:
~1 hr 18 mins
Color Coding (Caps) | Reagents | Description | GDQ6035-20R (20 tests) | GDQ6035-50R (50 tests) | GDQ6035-100R (100 tests) |
Red | 2X One-step Master Mix | Amplification Mix | 300 µL | 700 µL | 1.4 mL |
Amber | Primer-Probe Mix | Amplification Reagent | 40 µL | 100 µL | 200 µL |
Yellow | Positive Control (PC) | Positive Control | 20 µL | 50 µL | 100 µL |
Blue | Negative Control (NC) | Negative Control | 200 µL | 500 µL | 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. Storage condition is below -20 ˚C for no more than 3 days. Samples can be stored for a long time at -80˚C. Repeated freezing-thawing of samples 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 viral RNA extraction kit, and the extracted RNA could be directly used for detection. If the extracted RNA is not immediately tested after extraction, they can also be stored at -80°C and repeated freezing and thawing should be avoided. It is advised to start amplification with at RNA of high purity (O.D.260/280 = 2.0). A starting RNA concentration of at least 10 ng/ µL should be used. Too high RNA concentration might necessitate template dilution according to requirement.
*Note: Use of freshly extracted RNA is recommended.
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 PCR reagents should be kept on ice.
C. TaqMan qRT-PCR 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 PCR 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) |
5’ UTR region | FAM (Green) | 470 | 510 |
Reference gene (IC) | Cy5 (Red) | 625 | 660 |
E. PCR cycling conditions:
Step | Temperature (°C) | Time | No. of cycles |
Hold stage 1(RT-step) | 50 | 10 min | 1 |
Hold stage 2 | 95 | 3 min | 1 |
PCR Stage | 95 | 10 sec | 45 |
60 | 30 sec* | ||
*Acquisition step (Green/ Red Channel) |
F. Instrument compatibility:
The DiAGSure® Rhinovirus Infection Detection Kit (Multiplex, TaqMan based) is compatible 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, Quantstudio 5, BIORADTM CFX96, QIAGEN® Rotor Gene Q, Roche Light-cycler® 480.
G. Performance Evaluation:
I• Specificity:
The target sequences detected in this kit specifically targets the 5’-UTR gene of Rhinovirus. The primers used in the assay failed to give any amplification from RNA extracted from the sputum of healthy individuals eliminating the possibility of non-specific annealing with control human RNA.
Furthermore, the primers did not cross-react with viruses like Japanese encephalitis virus (JEV), West Nile virus, Chikungunya virus, Nipah virus, Hepatitis A virus, Hepatitis C virus, Hepatitis B virus Also, the primers did not cross-react with the most common bacteria like E. coli, B. subtilis, Pseudomonas sp. as well as pathogens like Mycobacterium tuberculosis, Streptococcus pneumoniae, Vibrio cholerae, Chlamydia trachomatis, and Salmonella typhi, and the protozoa Plasmodium sp. and Giardia lamblia.
II• Detection limit:
The absolute sensitivities were obtained using a dilution series of quantified synthetic RNA (in-vitro transcribed) as a template for detection. The Limit of Detection (LoD) was found to be 100 RNA copies/reaction under in-vitro conditions.
III• Safety information:
The DiAGSure® Rhinovirus Infection Detection Kit (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 | Cut-off Ct | Interpretation |
Viral gene – 5’UTR (FAM) | Ct≤40 | Viral RNA 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’s result 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 | Viral gene | IC | Interpretation | Results |
Fluorophore* | FAM/ Green | Cy5/Red | ||
Case-1 | Positive | Positive/ Negative | Rhinovirus Positive | Viral infection is detected in the clinical specimen. |
Case-2 | Negative | Positive | Rhinovirus Negative | Viral infection is not detected in the sample. |
Case-3 | Negative | Negative | Invalid | Testing should be repeated once from extraction. If a second failure occurs, it is reported to sender as invalid and sample recollection is recommended. |
* If the target gene signal (FAM Channel) is strong, the IC (Cy5 Channel) may be negative.
General precautions
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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