key: cord-0908143-ujf2vql5
authors: Yu, Xuelian; Ghildyal, Reena
title: Nested-RT-PCR and Multiplex RT-PCR for Diagnosis of Rhinovirus Infection in Clinical Samples
date: 2014-09-09
journal: Rhinoviruses
DOI: 10.1007/978-1-4939-1571-2_2
sha: 871e4ddd35c70372f5277d9da89a82895325883e
doc_id: 908143
cord_uid: ujf2vql5

Human rhinoviruses (HRVs) are positive-stranded RNA viruses belonging to the Enterovirus genus in the family of Picornaviridae. Identification of the specific strain in HRV disease has been difficult because the traditional serological method is insensitive, labor intensive, and cumbersome. With the fast progress in molecular biological technique, more sensitive and faster molecular methods have been developed, such as polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, and real-time RT-PCR. To improve the technique for defining the links between illnesses and specific strains of HRV, we developed RT-PCR specific for HRV as routine base. A multiplex RT-PCR that simultaneously identifies 12 respiratory viruses including HRV is also routinely used in our lab. Here we have described the specific steps of methods for identification of HRV from clinical samples, such as sample preparation, isolation of total RNA, nested-RT-PCR for HRV, Seeplex(®) RV15 ACE Detection method, gel electrophoresis, how to use the QIAxcel(®) capillary electrophoresis system, and results interpretation.

Human rhinoviruses (HRVs), members within genus Enterovirus in the family Picornaviridae , have a positive-strand RNA genome of 7,200 nucleotides covalently linked at the 5′ end to the viral protein 3B (VPg) and is translated cap-independently by internal ribosomal entry into a polyprotein (VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D), which yields the 11 proteins through various independently functioning intermediates, upon cleavage by viral proteases [ 1 ] . HRVs were originally classifi ed into two species, A and B, and approximately 100 serotypes [ 2 -4 ] . Advances in molecular methods led to the characterization of the third HRV group, a genetically heterogeneous third species, HRV species C(HRV-C) [ 5 , 6 ] . Identifi cation of the specifi c strain in HRV disease has been diffi cult because the traditional serological method is insensitive, labor intensive, and cumbersome [ 7 ] , which requires the isolation of HRV in susceptible cell cultures and neutralization tests against all 101 serotype-specifi c antisera [ 3 ] .

To improve the technique for defi ning the links between illnesses and specifi c strains of HRV, more sensitive and faster molecular methods have been developed. PCR, RT-PCR, and real-time RT-PCR represent the latest diagnostic methods that provide robust, reproducible results.

We present here an RT-PCR for identifi cation of HRVs and a multiplex RT-PCR that simultaneously identifi es 12 respiratory viruses; these assays are routinely used in our laboratory for diagnosis of HRV infection in clinical samples and have been optimized to enable simultaneous handling of large numbers of samples.

6. Electrophoresis apparatus. 7. UV transilluminator.

8. QIAxcel ® capillary electrophoresis system.

to microbiology laboratory for testing within 3 h of collection. Samples should be stored at 4 °C at this time ( see Note 4 ). For respiratory virus diagnostics two kinds of samples are collected, nasopharyngeal aspirates (NPA) and nasal swabs. NPAs require specialist equipment that must be handled by trained personnel; this procedure should be performed according to local safety and clinical guidelines. Nasal swabs are collected by inserting fl ocked tipped swabs into nostrils and must be performed by trained personnel only and according to local safety and clinical guidelines. Swabs must be inserted into tubes with VTM immediately after collection.

2. 1.5 ml sterile saline is added into each tube of NPA; tubes are centrifuged at 14,000 rpm (20,000 × g ) for 2 min; the supernatant is removed and pellet is resuspended in 200 µl of E-MEM, aliquoted, and kept at −70 °C. Nasal swabs are squeezed ten times against the tube walls in VTM and removed; tubes are centrifuged at 3,000 rpm (200 × g ) for 2 min; the supernatant is aliquoted and kept at −70 °C.

The protocol presented here is based on the QIAamp viral RNA isolation kit; any kit for isolation of total RNA from cell-free body fl uids may be used in its stead ( see Notes 2 and 7 ).

1. Equilibrate samples and Buffer AVE to room temperature (15-25 °C). 7. Carefully open the QIAamp spin column, and repeat step 6 .

8. Carefully open the QIAamp spin column, and add 500 μl of Buffer AW1. Close the cap, and centrifuge at 6,000 × g (8,000 rpm) for 1 min. Place the QIAamp spin column in a clean 2-ml collection tube, and discard the tube containing the fi ltrate.

9. Carefully open the QIAamp spin column, and add 500 μl of Buffer AW2. Close the cap and centrifuge at full speed (14,000 rpm; 20,000 × g ) for 3 min. Continue directly with step 10 , or to eliminate any chance of possible Buffer AW2 carryover, perform step 9(a) , and then continue with step 10 .

(a) (Optional): Place the QIAamp spin column in a new 2-ml collection tube (not provided), and discard the old collection tube with the fi ltrate. Centrifuge at full speed for 1 min.

10. Place the QIAamp spin column in a clean 1.5-ml microcentrifuge tube. Discard the old collection tube containing the fi ltrate. Carefully open the QIAamp spin column and add 60 μl of Buffer AVE equilibrated to room temperature. Close the cap, and incubate at room temperature for 1 min. Centrifuge at 6,000 × g (8,000 rpm) for 1 min. Viral RNA is stable for up to 1 year when stored at −20 °C or −70 °C.

We routinely perform reverse transcription using random hexamers and SuperScriptTM III Reverse Transcriptase Kit on GeneAmp PCR system 9700 according to the manufacturer's recommendations. Any other reverse transcriptase system can be

used, keeping in mind that some optimization may be required for optimal results ( see Note 9 ).

1. Briefl y centrifuge each reagent provided in the kit.

2. Prepare pre-mix 1 in a sterile, nuclease-free microcentrifuge tube as below, mix by brief vortex, and centrifuge briefl y:

Pre-mix 1 (for one reaction) 5 μg Total RNA 5 μl 50 ng/μl random primer 1 μl 10 mM dNTP mix 1 μl

DNase-and RNase-free water 3 μl

Calculate the required total amount of each reagent based on the number of reactions (samples + controls).

3. Label a 96-well plate, as the cDNA plate. Pipette 10 μl of premix into each well of the cDNA plate; cover with the sealing membrane and briefl y centrifuge the plate.

4. Place the cDNA plate into GeneAmp PCR system 9700, and incubate at 65 °C for 5 min. Take the cDNA plate out and put it on the ice. Centrifuge briefl y before removing the membrane seal.

5. Take another sterile, nuclease-free 1.5 ml microcentrifuge tube labeled as pre-mix 2. Add the following reagents (shown for one reaction); mix by quick vortex, and centrifuge briefl y.

10× RT buffer 2 μl

Total volume of pre-mix 2 10 μl

Calculate the required amount of each reagent based on the number of reactions (samples + controls).

6. Pipet 10 μl of pre-mix 2 into each well of the cDNA plate; cover it with the sealing membrane carefully and briefl y centrifuge the plate.

7. Place the cDNA plate into GeneAmp PCR system 9700, run the following cycling parameter: one cycle of (25 °C, 10 min); followed by one cycle of 50 °C, 50 min; then one cycle of (85 °C, 5 min) to stop the reaction. Take the plate out and put it on the ice. Centrifuge briefl y before removing the sealing membrane.

8. Pipet 1 μl of RNase H into each well of the cDNA plate; cover carefully with membrane seal and centrifuge briefl y.

9. Place the cDNA plate into GeneAmp PCR system 9700, and run one cycle of (37 °C, 20 min).

10. The fi rst-strand cDNA is synthesized and can serve as the template in the nested-PCR or the multiplex PCR or stored at −20 °C for future use.

This procedure consists of two PCR cycles, the fi rst run PCR using the cDNA as template and the second run PCR using the amplimers from the fi rst run PCR as template.

1. Prepare pre-mix 1 in the reagent preparation room ( see Note 8 ). Completely thaw and thoroughly vortex the buffer prior to use; then briefl y centrifuge each reagent.

2. In a sterile, nuclease-free microcentrifuge tube combine the following components to prepare the pre-mix 1 (shown for one reaction); mix by quick vortex and centrifuge briefl y. 1. Prepare pre-mix 2 in the reagent preparation room. Completely thaw and thoroughly vortex the buffer prior to use; then briefl y centrifuge each reagent.

2. In a sterile, nuclease-free microcentrifuge tube combine the following components to prepare the pre-mix 2 (shown for one reaction); mix by quick vortex and centrifuge briefl y. PCR products may be analyzed by agarose gel electrophoresis or by capillary electrophoresis. Figure 1 depicts a typical pattern obtained by gel electrophoresis, while Fig. 2 depicts a typical pattern obtained by capillary electrophoresis.

1. Separate the PCR products from the second run PCR by 2 % agarose gel electrophoresis and visualize by UV transillumination ( see Chapter 3 for detailed method).

(a) For reactions containing the 5× Green GoTaq™ Reaction Buffer, load samples onto the gel directly after amplifi cation.

(b) For reactions not containing any indicator dyes, add gelloading dye (e.g., bromophenol blue) before loading on the gel.

(c) Please see Fig. 1 for a picture of a sample gel of nested-PCR products from nasal swabs collected from children with suspected lower respiratory tract infection.

(d) There are single bright bands at the 110 bp size location of the positive control (PC), lanes labeled S5, S10, S21, and S32, but no bands around the same location in the negative control (NC), S1, S3, and S9 lanes. Fig. 1 Gel image of rhinovirus nested-RT-PCR products. The nasopharyngeal aspirate (NPA) samples (S) 1, 3, 5, 9, 10, 21, and 32 were collected from seven children with lower respiratory tract infection. Respiratory epithelial cells were collected from NPAs and processed for RNA isolation followed by nested-RT-PCR as described in Subheadings 2 and 3 . PCR products were separated on 2 % agarose gel electrophoresis and visualized by ethidium bromide staining; the relevant DNA marker is indicated 2. Capillary electrophoresis using QIAxcel system ( see Note 10 ). The 96-well plates can be run directly in the QIAxcel ® capillary electrophoresis system. Separation is performed in a capillary of a precast gel cartridge. Each sample is automatically loaded (according to voltage and time parameters) into an individual capillary and voltage is applied. As the molecules migrate through the capillary, they pass a detector that measures the fl uorescent signal. A photomultiplier detector converts the emission signal into electronic data, which are then transferred to the computer for further processing using BioCalculator software. After processing, the data are displayed as an electropherogram and a gel image. (c) When not in use, the 12-tube strip containing QX Alignment Marker should be stored at −20 °C. It should be equilibrated to operating temperature (20-25 ºC) and centrifuged briefl y before use.

Image of Seeplex RV 15 Multiplex products using QIAxcel capillary electrophoresis. NPA samples (S) 1-9 were collected from nine children with lower respiratory tract infection. NPA samples were processed for cell isolation, RNA extraction, and reverse transcription as in Fig. 1 . Seeplex RV 15 Multiplex PCR was performed as described in the text and PCR products separated by the QIAxcel ® Novel 12-channel capillary electrophoresis system. The reference table for RV15 ACE Detection (B set) marker is indicated (d) If the QIAxcel gel cartridge is being used for the fi rst time, intensity calibration should be performed ( see Note 11 ).

(e) Preparation of the gel cartridge: Add 10 ml QX Wash Buffer to both reservoirs of the QX Cartridge Stand (provided with the QIAxcel instrument) and cover with 3 ml mineral oil (supplied). Remove the QIAxcel gel cartridge from its packaging and carefully wipe off any soft gel debris from the capillary tips using a soft tissue. Remove the purge cap seal from the back of the QIAxcel gel cartridge and place it in the QX Cartridge Stand. Incubate new cartridges in the QX Cartridge Stand for 20 min prior to use. key can be inserted in either direction. Close the cartridge door. The cartridge ID, number of runs remaining, and cartridge type will be displayed automatically in the "Instrument Control" window once the smart key is latched. The system will not recognize the cartridge and will not operate if the smart key is not inserted. Add the DNA size marker (supplied with the kit) into the black well ( see Note 12 ). Load the 96-well plate containing the samples. The minimum sample volume required for analysis is 10 μl. Less than 0.1 μl of the sample will be loaded onto the QIAxcel gel cartridge for analysis. The remaining DNA can be kept for reanalysis or downstream processing. Results for nine nasal swab samples assayed by the Seeplex RV15 multiplex PCR products are shown in Fig. 2 .

According to the reference table provided by the manufacturer, samples 2 and 3 are probably positive for infl uenza A (a second specifi c PCR and/or sequencing of the PCR product needs to be performed to confi rm), sample 5 is positive for HRV, and sample 9 is positive for CoV-OC43. The size of samples 2 and 3 is equal to infl uenza A, the size of sample 5 is equal to human rhinovirus A/B/C, and the size of sample 9 is equal to human coronavirus OC43. Samples 4, 6, 7, and 8 are negative for all the respiratory viruses detected by Seeplex assay. 7. When the number of samples to be analyzed is less than 10, it is quicker and more convenient to isolate the total RNA from samples manually by using the QIAamp ® Viral RNA Mini Kit. When the number of samples is more than 10, it is laborious and time consuming to isolate the total RNA from samples manually and an automated system, e.g., the Roche MagnaPure LC 2.0, may be used.

8. Prepare the PCR pre-mix only in the pre-mix preparation room. Assay the fi rst-strand cDNA synthesis and perform template DNA/RNA or cDNA pipetting in the template room. Do not apply the fi rst-strand cDNA synthesis in the pre-mix preparation room to avoid contamination of the following PCR test. Ensure that the plates are covered and sealed with a membrane before they are transfered to PCR machine in the PCR amplifi cation room.

9. The 8-channel micropipette is preferred when using 96-well plates. It is less laborious and more accurate to aliquot reagent and pipet template DNA/RNA or cDNA into each well. Using multichannel micropipette also helps to reduce the contamination during PCR assay process.

10. The QIAxcel system offers a number of advantages over traditional slab-gel electrophoresis, including higher detection sensitivity, less sample wastage (minimal sample input volumes), fast analysis of up to 96 samples, and automated loading and analysis, making it the preferred method for analysis of DNA products when large numbers of samples are processed.

11. This step is not necessary if the QIAxcel gel cartridge has already been calibrated, unless it is being used on a different QIAxcel instrument or a different computer is used to operate the instrument. If a different computer is used, the calibration log fi le must be transferred from the computer used to operate the instrument to the new computer so that it is not necessary to run the calibration again. 

Picornavirus structure and multiplication in Virology

A cytopathogenic agent isolated from naval recruits with mild respiratory illnesses

A Collaborative report: Rhinovirusesextension of the numbering system from 89 to 100

A collaborative report: Rhinoviruses-extension of the numbering system

MassTag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness

Characterisation of a newly identifi ed human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis

Improved molecular identifi cation of enteroviruses by RT-PCR and amplicon sequencing

This work was supported by the Public Health Academic Leader Project (grant number GWDTR201201) from Shanghai Municipal Health Bureau.Nested-RT-PCR and Multiplex RT-PCR for Diagnosis…