3.1. Ethics Statement
In this study, four suspected of rabies patients with non-specific symptoms entered this study between April 2017 and April 2018 after obtaining the informed verbal consent. Also, all brains were samples remained from routine diagnostic activities at the National Center for Reference and Research on Rabies, Pasteur Institute of Iran and derived from animals suspected of rabies that had died. The center repository has been registered for research purposes in accordance with the license from the Iran Veterinary Organization.
3.2. Virus Isolates and Sample Collection
Rabies lyssavirus isolates 35009 (35009-RV-CVS, accession number. LT839616) (lethal dose 50%, LD50 > 10-8) obtained from the National Center for Reference and Research on Rabies, Pasteur Institute of Iran were included in this study. Also, specimens of the brain tissues preserved in the archive of the center were randomly selected so that the final results of the rabies test were 68% (34 cases) of the total positive population (50 cases). All brain tissue samples were previously diagnosed by dFA and MIT and then coded prior to qRT-PCR and heminested RT-PCR assays. All samples were stored at -70°C.
Moreover, three saliva samples at intervals of three to six hours from each of four suspected of rabies patients were collected for antemortem diagnosis of rabies. The histories of patients have been mentioned both specific and non-specific clinical symptoms such as irritability, headache, vomiting, mild fever, muscle aches, fatigue, retreat and in some cases photophobia. The main condition for entering the study was the positive results of the dFA test on their brain specimens after death that confirmed the presence of rabies virus infection in them. The saliva samples were collected from the oral cavity by a nurse on admission to the hospital via suction using a sterile syringe or a plastic dropper; while aspiration of sputum and throat is not acceptable. A healthy brain sample was also examined as the negative control. A brain sample that was positive in dFA was also considered to be the positive control.
3.3. Total RNA Extraction and Viral Complementary DNA (cDNA) Synthesis
Initially, 400 mL of each saliva samples was treated with 200 mL proteinase K and incubated for 30 minutes at 56°C; the samples were placed in a vortex every 5 to 10 minutes. Then total RNA of treated saliva and brain samples were isolated using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA was dissolved in the RNase free buffer provided with the kit. Total RNA was quantified at optical density (OD) 260 and 280 by Nanodrop 2000 spectrophotometer (Thermo Fisher, USA). A total of 50 to 100 ng viral RNA was converted into cDNA using reverse transcriptase polymerase chain reaction.
The RNA was subjected to cDNA synthesis using one µl of random hexamer (50 µM) and subjected to 70°C for five minutes and was later snap cooled on ice and briefly spun down. The RevertAid First Strand cDNA synthesis kit (Thermo scientific, Lithuania) was used for cDNA synthesis. Reverse transcriptase (Thermo scientific, Lithuania) mix was prepared and subjected to conditions 37°C for 60 minutes, 70°C for 5 minutes and chilling on ice for 5 minutes in a thermal cycler (Bio-Rad). The cDNA concentration was measured using nanodrop spectrophotometer (nanodrop technologies, CA) in ng/µL and quality was checked as a ratio of OD 260/280. The remaining parts of RNA and cDNA were frozen and stored at -70°C for further study.
3.4. PCR Amplification for Controlling of RNA Extraction Process
The quality of the RNA template was assessed by amplification of a housekeeping gene, β-actin, in each sample using PCR. Primers for the PCR assay were selected from Dacheux et al. study based on β-actin gene sequence in Genebank (accession No.: E00829) (12) and then were confirmed using the NCBI Nucleotide BLAST software (version 6.0) (Table 1).
Table 1.
The Primers for PCR Assay
Name | Length, bp | TM | Sequence | Position | Product, bp |
---|
β-actin-Taq1 | 25 | 64 | 73 | 5-TCACCCACACTGTGCCCATCTACGA-3 | 2206 | 295 |
β-actin-Taq2 | 25 | 66 | 74 | 5-CAGCGGAACCGCTCATTGCCAATGG-3 | 2500 |
Two µL cDNA was added to a 25 µL PCR mixture containing 12.5 µL 2× EasyTaq PCR SuperMix (+dye) (Yekta Tajhiz Azma, Iran) and 0.2 pM of each primer. The mixture was covered by 50 µL of light mineral oil. The initial denaturation temperature is 94°C for 3 minutes. The PCR program was 35 cycles (94°C, 30 seconds; 56°C, 45 seconds; 72°C, 40 seconds) and the final step was set at 72°C for 3 minutes using a DNA thermal cycler (Bio-Rad). The results were visualized under ultraviolet light by a transilluminator device following 1.5% agarose gel electrophoresis.
3.5. Development of Heminested RT-PCR
3.5.1. Primer Design for Heminested RT-PCR
Three primers were selected from Dacheux et al. study (12) based on conserved domains in RNA-dependent RNA polymerase (L) gene of RV strains PV (Genbank accession no. M13215) (13). The primers were confirmed using the NCBI Nucleotide BLAST software (version 6.0) (Table 2).
Table 2.
The Primers for Heminested RT-PCR Assay
Name | Length, bp | TM | Sequence | Position | Product, bp |
---|
Pvo5 | 20 | 35 | 64 | 5-ATGACAGACAAYYTGAACAA-3 | 7170 | First PCR: 320, second PCR: 250 |
Pvo8 | 22 | 40 | 69 | 5-GGTCTGATCTRTCWGARYAATA-3 | 7419 |
Pvo9 | 19 | 40 | 69 | 5-TGACCATTCCARCARGTNG-3 | 7489 |
3.5.2. Heminested RT-PCR Aassay
Two µL of cDNA was used in heminested RT-PCR. The reaction mixture contained 12.5 µL of Easy TaqPCR (2U) Supermix (+dye) (10 nM NTP) + (62/5 nM MgCl2), 0.2 pM primers individually, Pvo5 and Pvo9, and 9.5 µL of PCR water to a final volume of 25 µL. The amplification was done in the thermocycler (Bio-Rad) using thermal cycling and the conditions were as follow: one cycle of polymerase activation 94°C for 3 minutes, 35 cycles with denaturation at 94°C for 30 seconds, annealing at 56°C for 45 seconds, extension at 72°C for 40 seconds, and the final extension at 72°C for 3 minutes.
Two µL of the first stage PCR product was used in the second stage. The reaction mixture contained 12.5 µL of Easy TaqPCR (2U) Supermix (+dye) (10 nM NTP) + (62/5 nM MgCl2), 0.2 pM primers individually, Pvo5 and Pvo8, and 9.5 µL of PCR water to a final volume of 25 µL. The amplification was done in the thermocycler (Bio-Rad) using thermal cycling conditions as described previously. As the negative control, no template control (NTC) reaction was also used. At the end of the amplification program, the PCR products were analyzed by 1.5% agarose gel electrophoresis with SYBR Safe stain (Invitrogen).
3.6. Development of qRT- PCR
3.6.1. Designs of Primers and Probe for qRT- PCR
The TaqMan primers and a probe were designed by Oligo Analyzer software version 3.1 and silico software (Table 3). The primers and the probe for the detection of genotype 1 of rabies virus were designed based on the Pasteur virus (PV) L protein sequence available in the GenBank database (Genbank accession No. M13215). The TaqMan probe extracted from the Dacheux et al. paper (14) was labeled at the 5’ end with the FAM as a fluorophore dye and at a 3’ end with TAMRA as a quencher molecule.
Table 3.
The Primers and TaqMan Probe Sequences for qRT-PCR
Name Type | Length, bp | TM | nmol | Sequence | Sense | Position | Product, bp |
---|
Primer F | 26 | 63 | 25 | 5-TCTCTGTCCTAGATCAAGTGTTTGGA-3 | S | 7316 - 7341 | 98 |
Primer R | 29 | 64 | 25 | 5-GTCTGATCTGTCTGAATAATAGACCCAGG-3 | AS | 7385 - 7413 |
RV5 prob (FAM/TAMRA) | 32 | 70 | - | 5-AGRGTGTTTTCYAGRACWCAYGAGTTTTTYCA-3 | S | 7384 - 7353 |
3.6.2. qRT-PCR Assay
The qRT-PCR assay was performed using the Superscript III Platinum One-Step qRT-PCR (Invitrogen) kit. The mixture contained of 5 µL 2 × reaction mix, 0.2 µL Superscript III RT/Platinum TagMix, 0.25 µL MgsO4 (50 Mm), 0.2 µL RNasin, 0.025 µL ROX, 0.15 µM RV5 probe and 0.5 µL of a 10 mM solution of sense/anti-sense primers, 0.5 µg of template cDNA added with H2O to a total of 10 µL. We used the negative control and and NTC reaction along with other samples. Thermal cycling conditions were carried out by hold one stage at 45°C for 15 minutes and hold two stages at 95°C for 3 minutes followed by 40 cycles at 95°C for 15 seconds and 61°C for 1 minute. All tests were performed in triplicate.
3.7. Evaluation of the Clinical Sensitivity and Specificity of the qRT-PCR Assay and Heminested RT-PCR Compared with FAT
Finally, we evaluated the qRT-PCR and heminested RT-PCR assays by dFA, on 50 samples obtained randomly from the repository of the National Center for Reference and Research on Rabies, Pasteur Institute of Iran. The clinical sensitivity and specificity of qRT-PCR and heminested RT-PCR methods were evaluated using the below formulae.
Equation 1.
Equation 2.
The qRT-PCR assay was also carried on total RNA extracted of saliva samples of four patients who had the conditions for entering the study as described above.
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