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FULL LENGTH Iranian Biomedical Journal 24 (6): 399-404 November 2020

A Real-Time RT-PCR Assay for Genotyping of Rotavirus

Seyed Dawood Mousavi-Nasab1,2, Farzaneh Sabahi2, Hooman Kaghazian1, Mahdi Paryan1,


Siamak Mirab Samiee3, Mostafa Ghaderi4, Fatemeh Zali5 and Manoochehr Makvandi6*
1
Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran,
2 3
Iran; Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran; Reference
4
Health Laboratories Research Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran; Department
5
of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran; Department of Clinical Biochemistry,
6
Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran; Infectious and Tropical Diseases
Research Center, Health research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Received 14 July 2019; accepted 26 December 2019; published online 13 June 2020

ABSTRACT

Background: HRV is the causative agent of severe gastroenteritis in children and responsible for two million
hospitalizations and more than a half-million deaths annually. Sequence characteristics of the gene segments
encoding the VP7 and VP4 proteins are used for the genotype classification of rotavirus. A wide variety of
molecular methods are available, mainly based on reverse transcription PCR for rapid, specific and sensitive
genotyping of rotaviruses. This study describes an alternative real-time PCR assay for genotyping of rotavirus.
Methods: The samples of stools studied in this research have been collected from patients referred to Children's
Medical Centers, Tehran, Iran. Rotavirus detection and genotyping were performed using the RT-PCR and semi-
nested RT-PCR, respectively. Samples were then genotyped with a new real-time PCR. Results: The real-time PCR
was able to genotype all positive samples with a mean Ct of 28.2. Besides, a concordance rate of 100% was
detected between real-time PCR and semi-nested RT-PCR. Conclusion: In this study, the genotyping of rotavirus
with real-time PCR showed that this method can provide several favorable features, including high sensitivity and
specificity, and a wide dynamic range for rotavirus genotyping. DOI: 10.29252/ibj.24.6.394

Keywords: Gastroenteritis, Genotype, Real-time polymerase chain reaction, Rotavirus

Corresponding Author: Manoochehr Makvandi


Infectious and Tropical Diseases Research Center, Health research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; Tel.:
(+98-613) 3354389; Fax: (+98-613) 3361544; E-mail: manoochehrmakvandi29@yahoo.com

INTRODUCTION and are classified as a separate genus (rotavirus) within


the family Reoviridae[4]. Rotavirus genome is

H
uman rotavirus is the most common cause of composed of 11 double-strand RNA segments, which
severe gastroenteritis in infants and young are surrounded by the inner capsid proteins, including
children under the age of five years VP1, VP2, and VP3[5]. VP6 proteins form the middle
worldwide, accounting for two million hospitalizations layer of the virus capsid, and VP4 and VP7 proteins
and more than a half-million death every year[1,2]. constitute the outer layer[6]. The Rotaviruses are
Rotavirus is known to be transmitted person to person currently categorized into eight groups, A through H,
by the fecal-oral route. In developing countries, its according to the group- and subgroup-specific antigens
transmission occurs through contaminated water. Base located at the VP6 region[7]. Four serogroups of
on reports, rotavirus can spread from child to child via rotavirus, including A, B, C, and H, are recognized to
the contamination of hands[3]. Rotaviruses are naked be human pathogens. Rotavirus group A is responsible
double-strand RNA viruses with a segmented genome for more than 90% of all cases[4]. Sequence

List of Abbreviations:
FBS, fetal bovine serum; miRNA, microRNA; CCND1, Cyclin D1; GFP, green fluorescent protein; HRV, human rotavirus; IC50, 50%
inhibitory concentration
Real-Time RT-PCR for Genotyping Mousavi-Nasab et al.

characteristics of the segments encoding the VP7 [G, minutes. Rotavirus RNA was extracted from 100 µl of
glycoprotein] and VP4 [P, protease-sensitive] are used the filtered supernatant by a standard phenol–chlorform
for serotype and genotype classification. To date, extraction method[20].
group A rotaviruses have been grouped into at least 27
G and 37 P genotypes based on the differences in their Reverse transcription
VP7 and VP4 gene sequences, respectively. Among The cDNA was synthesized with the RevertAid RT
them, newly 12 G and 15 P genotypes are thought to Reverse Transcription Kit (Thermo Fisher Scientific,
infect humans[8,9]. G1, G2, G4, G9, and G non-type USA) according to the manufacturer’s instructions.
were shown to be the most prevalent G type, while P Briefly, reverse transcription was carried out in a final
[8], P [4], and P non-type were found to be the most volume of 20 μl containing 4 μl of 5× reverse
frequent P type in Iran, respectively[10-12]. transcription buffer, 1 μl of 10 mM dNTPs, 1 μl of 0.2
Rotavirus classification methods have evolved U/μl random hexamer, 1 μl of 40 U/μl RNase inhibitor,
primarily from antibody-based assays to genetic 1 μl of 200 U/μl reverse transcriptase enzyme, 8 μl of
characterization[13]. Sequencing and phylogenetic diethyl pyrocarbonate (RNase-free water), and 4 μl of
analyses are currently considered to be the gold the extracted RNA. The tubes were incubated at 42 ºC
standard methods for HRV genotyping[7,14,15]. for 1 hour.
Nowadays, a wide variety of molecular methods,
including Southern blot, Northern blot, reverse line RT-PCR for rotavirus detection
blot hybridization, PCR-ELISA, and RFLP, have been PCR amplification was carried out in a final volume
developed for rapid, specific and sensitive genotyping of 25 µl containing 2.5 μL of the 10× PCR buffer
of rotaviruses[16,17]. Multiplex semi-nested RT-PCR has (CinnaGen, Iran), 1 μL of each 10 pmol/μL primer, 1
been the primary rotavirus genotyping method μL of 10 mM dNTPs (Fermentas UAB, Lituania), 1 μL
discriminated by gel electrophoresis based on the of 500 μ/μl of Taq DNA polymerase (CinnaGen), 1.5
amplicon length. Nearly all studies carried out in Iran μL of 50 mM of MgCl2 (CinnaGen), 12 μL of H2O, and
have used multiplex semi-nested RT-PCR for 4 µl of the cDNA template. Two primers (forward: 5′-
genotyping HRV. Using high-throughput real-time GAC GGV GCR ACT ACA TGG T-3′ and reverse: 5′-
PCR-based genotyping for rotaviruses has substantially GTC CAA TTC ATN CCT GGT G -3′) were used to
reduced the risk of cross-contamination, resulting in amplify the VP6 fragment[21]. The PCR conditions
faster turn-around time and higher sensitivity as were set under the following conditions: an initial
compared with the conventional multiplex semi-nested denaturation at 95 °C for 5 min, 40 cycles, including
RT-PCR[18,19]. denaturation at 94 °C for 1 min, annealing at 55 °C for
The purpose of this study was to investigate the 1 min, elongation at 72 °C for 1 min, and a final
potential value of a real-time PCR method for simple extension step at 72 °C for 10 min. Amplifications
and fast genotyping of HRVs using Iranian strains. In were performed using the Verity™ 96-well Thermal
the present study, we attempted to perform the analysis Cycler (Applied Biosystems, Foster City, CA, USA).
of a TaqMan real-time PCR assay that, to the best of
our knowledge, has not yet been used for typing of Multiplex semi-nested RT-PCR
HRV infections in Iran. G and P typing of HRV-positive samples were
obtained by multiplex semi-nested RT-PCR assays
using both consensus and type-specific primers, as
MATERIALS AND METHODS described previously[20]. The PCR products were
analyzed on a 2% agarose gel and visualized using
Sample collection and processing GelRed dye (GelRedΤΜ Nucleic Acid Gel Stain).
A total of 120 stool samples were obtained from
children aged five years and younger with a primary Real-time PCR for rotavirus genotyping
diagnosis of acute non-bloody gastroenteritis who Recently, Kottaridi et al.[21] developed two panels of
refereed to Children’s Medical Center in Tehran, Iran, real-time RT-PCR assays for the detection of G1-G4
from May 2013 to May 2014. Criteria for collecting and G9, P [4], and P [8]. In our study, based on
these samples were the absence of leukocytes, red multiplex semi-nested RT-PCR result, the genotype-
blood cells and pus in the stool. The stools stored at - specific primers and probes were adapted or
70 °C after primary analysis[4]. modified[16,21]. Both probes and primers were evaluated
A 10% (w/v) suspension of each stool sample was separately for each genotype based on the most
prepared for RNA extraction. Briefly, one gram (pea- conserved region, using the Allele ID® (PREMIER
sized) or 100 μl of each stool sample was dissolved in Biosoft International) and ClustalW tools. In some
1000 µl of PBS and then centrifuged at 1500 ×g for 20 cases, degenerate nucleotides were designed to ensure

400 Iran. Biomed. J. 24 (6): 399-404


Mousavi-Nasab et al. Real-Time RT-PCR for Genotyping

the amplification of all HRV genotypes. The Evaluation of real-time PCR performance
oligonucleotides primers and probes were synthesized The PCR products from each genotype were purified
by Macrogen (Macrogen, South Korea). The six primer using the QIAquick PCR Clean-up Kit (QIAGEN,
pairs, along with six probes labeled with different Hilden, Germany) and then cloned into the pTZ57R/T
fluorophores, were used to amplify and detect vector using the InsTAclone PCR cloning kit (Thermo
genotypes G1, G2, G9, P4, P8, and the internal control Fisher Scientific). The plasmid was extracted using the
(RNase P)[21,22]. The details of primers, probes and DNA-spin Plasmid DNA Purification Kit (iNtRON
fluorophore/quenchers are shown in Table 1. To Biotechnology, South Korea). Plasmid concentration
facilitate the use of real-time PCR and to enhance the was determined by measuring UV absorbance at 260
sensitivity of the assay, three real-time PCR panels nm. The plasmid was then diluted in a Tris-EDTA
were formulated in this study, in which the panel I was buffer. Relative sensitivity and lower limit of detection
designed for the detection of G1 and G2 genotypes, the of the assay determine the base of tenfold serial
panel II for the detection of G9 type as well Rnase P as dilutions of each plasmid.
internal control assay, and panel III for the To assess the specificity and possible false-positive
identification of P [4] and P [8] genotypes. detection by the genotype-specific primer-probe sets,
Real-time PCR genotyping was carried out in a the positive samples for human adenovirus, norovirus,
LightCycler™96 system (Roche, Basel, Switzerland) sapovirus, Escherichia coli, campylobacter,
Kottaridi et al.[21] reported various annealing Cryptosporidium, and Giardia lamblia were
temperatures [55–65 °C] with the final concentrations investigated in this study. Additionally, partial
range of 200 to 600 nmol for primers and probes. The sequencing was performed for 12 out of the 28
real-time PCR reactions were performed in a 25-µl specimens to verify the accuracy of HRV genotyping
final volume, containing 2 µl of the cDNA template, by real-time PCR. Analysis of sequencing was blasted
400 nM of each primer, and 200 nM of each probe. to determine the nucleotide identity (NCBI database)
The Ct value was then determined. The amplification and further analysis was performed using
was performed under the following thermal conditions: MEGA version 6[23].
initial denaturation at 95 ºC for 5 min, 40 cycles of
denaturation at 95 ºC for 15 s, annealing at 60 ºC for 30 Statistical analysis
s, elongation at 72 ºC for 30 s, and a final extension Comparison test was used to assess the consistency
step at 72 °C for 5 min. Based on the Ct value, the of real-time PCR and multiplex semi-nested RT-PCR
optimal conditions were determined, first for a single results. A p value of less than 0.05 was considered to
reaction and then for a set of multiple panels targeting be statistically significant.
G1/G2, G9/RNase P, and P4/P8 types.

Table 1. Primers and probes used for rotavirus genotyping by real-time PCR
Genotype Primer sequences Probe sequences Fluorophore/
quencher
G1F AGCTGATTTGATATTGAATGAATGG
G1R CACAGTACAYGATGATCCCATTG TCCACTTATTYGATTCTCCCGATTGYT FAM/BHQ1

G2F ACATTTGAGATTGTTGCVTCGTCTG
G2R TGGAACTGTYGTTGGATCAGCAG AGTGCRTTCGGTCCACCAACTTGAA HEX/BHQ1

G9F ACTTGATGTDACTACAAATACCTG
ATCTAACACATCTGAGCCACCGACTTG HEX/BHQ1
G9R TGTGGTGYAGTAGTTGGATCYG

P4F TGAYGAAATAGARCAGATTGGATC
P4R CCATCTAAAAYTGGTTCCACTG AATCTCTCCGTGTCCCCAATYRACTG FAM/BHQ1

P8F TAGACGTACACTAACTTCTGATAC
P8R TTGARCTATCRGTAGTAGCC CACCATGAAATGTCCATATTCTTCCACC HEX/BHQ1

RNase PF AGA TTT GGA CCT GCG AGC G


RNase PR GAG CGG CTG TCT CCA CAA GT TTCTGA CCTGAA GGCTCT GCG CG FAM/BHQ1
F, forward; R, reverse

Iran. Biomed. J. 24 (6): 399-404 401


Real-Time RT-PCR for Genotyping Mousavi-Nasab et al.

Ethical statement DISCUSSION


The above-mentioned sampling protocols were
approved by Tarbiat Modares University, Tehran, Iran The severe gastroenteritis due to HRV occurs
(ethical code: 52/5140). A verbal consent and interview annually throughout the world, especially in
were taken from either parents of the enrolled child developing countries, with high morbidity and
prior collection of stool samples. mortality rates. The prevalence of different rotavirus
genotypes varies in different geographical areas[24,25].
Interestingly, by using multiplex nested RT-PCR, the
RESULTS G1, G2, G9, mixed G1/G2, P4, and P8 genotypes were
detected for VP7 and VP4; however, we did not detect
In the present study, 28 (23.3%) patients were found some of the genotypes, such as G4 and G8[10,11]. The
to be positive for HRV infection by RT-PCR method absence of these genotypes in our study is probably
based on VP6 gene of rotavirus. The G and P due to the geographical sampling bias. Although
genotypes were detected in 28/120 patients multiplex semi-nested RT-PCR assay has been
by multiplex semi-nested RT-PCR. Overall, G1 recommended by WHO for HRV genotyping, this
(75%) was found to be the most dominant genotype, method is slow and prone to contamination. Moreover,
followed by G2 (14.3%), G9 (7.14%), and the turn-around time for multiplex semi-nested RT-
mixed G1/G2 (3.58%). Moreover, in terms of p PCR assay is higher than real-time PCR assey[20,21].
genotypes, P8 (75%) and P4 (25%) were dominant. The application of real-time PCR is generally regarded
Surprisingly, the G4 and G8 genotypes were not as a diagnostic tool since it provides quick, quantitative
detected. and reliable measurements. Furthermore, because real-
time PCR-based genotyping methods were proved to
Performance criteria for real-time PCR genotyping be significantly cost-effective, this technique can serve
Accuracy and specificity as an alternative tool to the multiplex semi-nested RT-
Accuracy of RNA extraction and amplification were PCR method. Kottaridi et al.[21] was applied the real-
confirmed by the production of a signal from the time PCR as a diagnostic tool for rotavirus genotyping,
RNase P internal control. There were no false-positive but in developing countries, because of genetic
or false-negative results among the patients with the diversity and co-infection with other gastrointestinal
expected VP7 and VP4 genotypes. Regarding the pathogens, it is required to re-evaluate molecular
isolated HRVs, no cross-reactivity was observed for techniques for the determination of rotavirus
adenovirus, strovirus, norovirus GI, norovirus GII, genotypes[26,27]. Therefore, these issues highlight the
sapovirus, Escherichia coli, campylobacter, need for the analysis of a method that would allow the
cryptosporidium, and Giardia lamblia. The results cost-effective, rapid and highly sensitive detection of
obtained by real-time PCR genotyping and partial HRV genotypes with a wider dynamic range. To the
sequencing of the amplified product showed 100% best of our knowledge, this is the first time that the
concordance to the both assays. real-time PCR assay has been utilized for the detection
of HRV genotypes in Iran. For this purpose, the real-
Correlation between real-time PCR and multiplex time PCR used to determine the G-P assay of rotavirus
semi-nested RT-PCR with TaqMan probes.
Both real-time PCR and multiplex semi-nested RT- The results of our TaqMan RT-PCR assay showed
PCR were able to detect all the 28 (100%), and the high accuracy and excellent correlation with
mean Ct value was 28.2. conventional multiplex semi-nested RT-PCR and
sequencing. The presented method also demonstrated
Limit of detection high specificity and no cross-reaction with the above-
To evaluate real-time PCR efficiency, standard
curves were constructed using serial dilutions (from Table 2. Real-time PCR performance for rotavirus
10-1 to 10-10) each plasmid. The lowest average of 10 genotyping
copies per reaction could detect 100% of all known Genotype
r2
Average
HVR genotypes, which have been identified in this panel efficiency (%)
study. The panel targeting the mixed G1/G2 genotypes G1/G2 ≥0.94 98
yielded an r2 ≥ 0.94, while the panel targeting G9/RNase P ≥0.99 97
G9/RNase P and P4/P8 provided an r2 ≥ 0.99 for the P4/P8 ≥0.99 95
standard curve (Table 2 and Fig. 1)

402 Iran. Biomed. J. 24 (6): 399-404


Mousavi-Nasab et al. Real-Time RT-PCR for Genotyping

Fig. 1. Limit of detection and real-time PCR performance for rotavirus P4 genotyping on LightCycler 96 system.

mentioned pathogens. This assay with a lower limit of 136-141.


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6. Malik J, Bhan MK, Ray P. Natural immunity to
rotavirus infection in children. Indian journal of
This project was partially supported under the grant biochemistry and biophysics 2008; 45(4): 219-228.
number U-93050 by Deputy Research, Ahvaz 7. Matthijnssens J, Ciarlet M, McDonald SM, Attoui H,
Jundishapur University of Medical Sciences, and Bányai K, Brister JR, Buesa J, Esonna MD, Estes Mk,
Ahvaz, Iran. Gentsch JR, Itueeiza-Gómara M, Johne R, Kirkwood
CD, Martella PP, Nakagomi O, Parreno V, Rahman M,
CONFLICT OF INTEREST. None declared. Ruqqeri FM, Saif LJ, Santos N, Stever A, Taniquuchi
K, Patton JT, Desseelberger U, Van Ranst M.
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