A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus

Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.

outbreaks. Having a right tool for rapid and specific detection of GETV infection, thus, is very important. Virus isolation and serological tests, for instance serum neutralization test, are commonly performed for the diagnosis of GETV infection 17,22,26 . However, these methods are laborious and time-consuming. Furthermore, the serological methods can be challenging due to the possible cross-reactivity between other alphaviruses. Various reverse-transcription polymerase chain reaction (RT-PCR) assay have been developed and used for detection of GETV RNA genome [27][28][29] . However, an assay for both detection and quantification of GETV is still limited in the present. Hence, in this study, we intended to develop a quantitative RT-PCR (qRT-PCR) assay for rapid detection and quantification of GETV.

Results
Design of GETV-specific qRT-PCR assay primers and TaqMan probe. Two sets of GETV-specific primers and TaqMan minor groove binder (MGB) probes were designed based on the alignment of the conserved nsP1 and nsP2 gene region of 23 global GETV genome, respectively (Table 1 and Fig. 1). Both sets of qRT-PCR primer and probes detected the two GETV strains (MM2021 and B254) used in this study ( Supplementary  Fig. S1).  Fig. S2).
Evaluation of qRT-PCR assay. The qRT-PCR assay using nsP2 primers and probe for detection of GETV RNA was evaluated by testing on a total of 16 simulated clinical samples containing GETV titers ranging from 1 × 10 5 to 1 × 10 0 plaque forming unit per ml (PFU/ml). The qRT-PCR assay detected up to as few as 1 PFU GETV in the simulated clinical samples. The qRT-PCR assay detected GETV RNA in all of the positive samples and none of the GETV negative samples showed positive amplification (Supplementary Table S1). The Diagnostic Test Calculator by Evidence-Based Medicine Toolbox calculated a sensitivity of 100% (95% CI = 78.5-100%) and specificity of 100% (95% CI = 34.2-100%) for the GETV qRT-PCR assay developed in this study ( Table 2).

Discussion
In the present study, two TaqMan MGB probe-based qRT-PCR assays targeting nsP1 and nsP2 genes of GETV were developed. The findings showed that the assay using nsP2 primers and probe was better for the detection and quantification of GETV RNA as it could detect all the GETV strains used in this study without cross-reacting with other common arboviruses including CHIKV, SINV, DENV-1, DENV-2, DENV-3, DENV-4, ZIKV P6-740, ZIKV MR766, JEV, and LGT. Probit analysis revealed that the detection limit of the qRT-PCR assay at the 95% probability level was 63 copies RNA. The qRT-PCR assay was sensitive and specific for the detection of GETV in all tested simulated clinical samples. The qRT-PCR assay developed here used a TaqMan MGB probe, which gives an advantage of increased specificity and lesser background noise in comparison to the conventional TaqMan probe 30,31 . In this study, the GETV qRT-PCR primers and probe were designed based on the non-structural protein gene sequences due to their highly conserved nature in the genome yet contain enough genetic variations to differentiate from the other alphaviruses. Several previous studies have reported conventional single-plex and multiplex RT-PCR assays for detection of GETV RNA using primers targeting nsP1, capsid and E1 gene 27,29,32 . Recently, Shi and the group reported a TaqMan probe-based qRT-PCR assay for detection of GETV RNA 32 . The primers designed targeted conserved regions of nsP1 gene of GETV. However, the specificity of the primers was not tested against other closely related alphaviruses or flaviviruses but other livestock viruses. In this study, the GETV nsP1 primers and probe showed cross-reactivity with CHIKV, SINV and DENV, although the in silico study of the primers showed mismatches with the sequences of other alphaviruses. In contrary, the qRT-PCR assay using GETV nsP2-spesific primers and probe showed better sensitivity without cross-reacting with other closely related arboviruses that share a mosquito vector with GETV. Thus, this assay is specific for detecting GETV and important for virus surveillance in mosquitoes. The qRT-PCR assays developed in this study detected up to as few as 10 copies of GETV RNA. The detection limit of the nsP1-and nsP2-specific primers and probes at the 95% probability level was determined to be 195 and 63 copies of viral RNA, respectively. Shi et al. has reported that the detection limit of their qRT-PCR assay was 25.5 copies RNA, which is at the same log scale as our assay using the nsP2-specific primers and probe. The information on the confidence level of the qRT-PCR assay of Shi et al., however, was not available.
In order to quantify GETV RNA in the samples, a panel of GETV RNA standard with known copy number is needed. Our GETV RNA standard were prepared using RNA in vitro transcribed from a plasmid vector carrying the nsP1 or nsP2 target sequences of GETV. The RNA copy number was calculated using formula based on the weight and sequence length of RNA transcript. In order to ensure the accuracy of RNA copy number, the RNA standard must be properly in vitro-transcribed. Linearization of the plasmid by single restriction enzyme digestion was performed before the in vitro transcription, this is to ensure that the RNA standard copies produced were all in the same length, which in turn enable the accurate calculation of the RNA copy number.
Considering the increasing GETV infection threats pose to animals, especially horses and pigs, leading to outbreaks in Japan and China 16,18,19,22 , a proper early detection tool in monitoring GETV activities in Malaysia is highly needed. In 1960s, the antibody against GETV has been frequently found in the horses and pigs in  Peninsular Malaysia 11 and yet to date the disease in the animals has not been reported. This could be due to the under-reporting and under-diagnosis because of unavailability of proper detection tools. Nevertheless, the following studies on GETV in the country are especially lacking. In Japan, GETV has been isolated from a range of animal samples including serum, saliva and nasal swab samples 23 . In this study, simulated clinical samples were used to assess the sensitivity and specificity of the qRT-PCR assay due to the lacking of well-characterized clinical samples of GETV in the region. The simulated clinical samples were prepared by spiking various concentrations of GETV into healthy human serum and saliva. The qRT-PCR developed in this study detected the GETV at titer of as few as one PFU/mL in both the simulated serum and saliva specimens. The findings showed that the qRT-PCR was sensitive enough to diagnose those infected animals which were still infectious. Early detection of the infected animal during the viremia phase allows immediate implementation of the disease control measures such as insecticide fogging and quarantine of the infected animals from subsequent mosquito bites, which in turn could reduce the transmission of the GETV. The discrepancy in the GETV RNA copies detected in serum and saliva samples, however, may indicate the differences in limit of detection of the qRT-PCR assay in different human matrices. Due to the small number of simulated clinical samples used in this study, the values for specificity, sensitivity, positive predictive value and negative predictive value need to be taken with caution. Further evaluation of the qRT-PCR in the field setting with a larger sample size of actual clinical samples and field-caught mosquitoes is warranted in future.
In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV. The qRT-PCR assay would be useful not only for rapid diagnosis of GETV infection and virus surveillance but also for the research applications.

Simulated clinical samples. The study obtained ethics approval from the UMMC Medical Research Ethics
Committee (MREC ID No: 2017220-4938). All methods were carried out in accordance with relevant guidelines and regulations. All simulated clinical samples were prepared by spiking GETV into actual human saliva and serum obtained from a healthy volunteer with informed consent at final concentrations ranging from 1 × 10 5 to 1 × 10 0 PFU/ml. Serum and saliva spiked with serum free medium acted as negative samples.

RNA extraction.
Total RNA was extracted from 140 µl of infected cell culture supernatant or simulated clinical samples using QIAamp Viral RNA Mini Kit (Qiagen, Germany) following the manufacturer's protocol. The RNA was eluted in 60 µl of nuclease-free water and stored at −80 °C until needed.
Design of GETV-specific qRT-PCR assay primers and TaqMan probe. A total of 23 GETV complete genome sequences were retrieved from GenBank (Supplementary Table S2). Multiple sequence alignment was performed using Clustal X 2.0. The complete genomes of other alphaviruses were also included during the primer design. Two sets of GETV-specific primers and TaqMan MGB probes were designed based on the conserved regions of nsP1 and nsP2 genes, respectively, using Primer Express (Version 3.0.1) and GeneDoc (Version 2.7). The pairs of primers and probes were analysed based on the penalty points calculated by the Primer Express software. The GC content, melting temperature, presence of dimers and hairpin formation were further examined for each primer and probe using the IDT's OligoAnalyzer Tool. The primers and probes were synthesized by Applied Biosystems ™ . The coverage of the qRT-PCR primers and probe was validated by evaluating the assay using viral RNA extracted from the different GETV strains.
Preparation of GETV RNA standard. Two GETV RNA standards containing the nsP1 and nsP2 target sequences of GETV strain MM2021, respectively, were generated and amplified by RT-PCR using primer pairs designed using GeneDoc software (Supplementary Table S3). The RT-PCR reaction consist of 25 µL of 2x MyTaq One Step Mix, 0.5 µL of reverse transcriptase, 1.2 pmol of each forward primer and reverse primer, 1 µL of RiboSafe RNase inhibitor, 2 µL of RNA template and 19.5 µL of nuclease-free water. The RT-PCR was performed using Applied Biosystems Veriti Thermal Cycler according to the following condition: 20 min at 45 °C, 1 min at 95 C followed by 40 cycles of amplification (10 s at 95 °C, 10 s at 60 °C, 30 s at 72 °C). The nsP1 and nsP2 amplicons were ligated into two pGEM ® -T Vectors respectively, using T4 DNA Ligase (Promega, USA) according to manufacturer's protocol. The reaction was incubated for 1 hour at room temperature. The recombinant plasmids were linearized by restriction enzyme digestion using BamHI (Promega, USA). The GETV RNA standards were in vitro transcribed from the linearized recombinant pGEM-T plasmids ( Supplementary Fig. S3) using the MEGAscript TM T7 transcription kit according to manufacturer's protocol. The in vitro transcription was performed at 37 °C for at least 2 hr. DNase treatment and lithium chloride precipitation steps were also performed according to manufacturer's protocol to purify the in vitro transcribed RNA. The purity and concentration of RNA was measured by spectrophotometer at 260 nm. The RNA copy number (copies/µL) was calculated based on the sequence length and weight of GETV RNA by using the web-based ENDMEMO DNA/RNA Copy Number Calculator (http://www.endmemo.com/bio/dnacopynum.php).
Detection limit of qRT-PCR assay. The detection limit of the qRT-PCR assay using the primers and probes specific to GETV nsP1 and nsP2 genes was assessed using a panel of respective serially diluted in vitro transcribed GETV RNA standard (10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 50, and 10 copy numbers). The detection limit test of qRT-PCR was repeated for at least four times for standard samples with less than 10 7 RNA copies. The qRT-PCR was performed in a final volume of 15 μl containing 3.75 μl of 4× Coverage and cross-reactivity of qRT-PCR assay. The coverage and cross-reactivity of the primers and probes were tested by evaluating the qRT-PCR assay on viral RNA extracted from two GETV strains (Malaysian strains: M2021 and B254), CHIKV, SINV, DENV-1, DENV-2, DENV-3, DENV-4, Asian ZIKV P6-740, African ZIKV MR766, JEV and LGTV. All of these viruses were obtained from the TIDREC viral repository.
Evaluation of qRT-PCR assay. The sensitivity and specificity of the qRT-PCR assay for detection of GETV RNA was assessed using the simulated clinical samples as previously described. Serum and saliva samples were obtained from healthy volunteer and were spiked with a panel of 10-fold serial diluted GETV to make final concentrations ranged from 1 × 10 5 to 1 × 10 0 PFU/ml. In brief, 20 µL of 1 × 10 6 , 1 × 10 5 , 1 × 10 4 , 1 × 10 3 , 5 × 10 2 , 1 × 10 2 , 1 × 10 1 PFU/ml GETV were added into 180 µl of serum or saliva samples. Serum sample and saliva sample spiked in with serum free media only acted as negative control.
Statistical Analysis. All statistical analyses were performed using IBM SPSS Statistics, version 21 (IBM Corporation, New York, United States). A probit analysis was performed to calculate the detection limit of the qRT-PCR assay at a 95% probability level. The sensitivity and specificity of qRT-PCR assay were calculated using web-based EBM Diagnostic Test Calculator (http://ebm-tools.knowledgetranslation.net/calculator/diagnostic/).