doi:10.5455/vetworld.2013.189-192 Sensitivity comparison of nested RT-PCR and TaqMan real time PCR for intravitam diagnosis of rabies in animals from urine samples

Aim: Early diagnosis of dreadful rabies is of utmost importance to restrict number of contacts and timely administration of post exposure prophylaxis. The present study was conducted to evaluate the sensitivity comparison of Nested RT-PCR with TaqMan real time PCR technique for intravitam diagnosis of rabies in animals from urine samples. 
 
Materials and Methods: Advance molecular approaches Nested RT-PCR and TaqMan real time PCR was employed on 21 urine samples for intravitam diagnosis of rabies. Comparison of both the techniques was done with standard immunofluorescence test (FAT) applied on brain for postmortem confirmation of rabies. 
 
Result: Rabies viral RNA was detected in 6/21 (28.57%) and 11/21 (52.38%) urine samples by application of Nested RT-PCR and TaqMan real time PCR respectively. Sensitivity obtained from both the techniques was 62.50% and 78.94% respectively when compared with gold standard immunofluorescence test (FAT). 
 
Conclusion: TaqMan real time PCR can serve as more sensitive and viable approach for the intravitam diagnosis of rabies as compared to Nested RT-PCR for detection of rabies from urine of suspected animals. 
 
Clinical importance: This study may serve as background for future intravitam rabies diagnostics.


Introduction
Urine samples were collected from 21 rabies suspected animals (14 buffaloes, 4 cattle's and 3 dogs) presented Since time immemorial rabies continues to be a to the Veterinary Clinics, GADVASU, Ludhiana, major health threat to mankind as well as all warm blooded Punjab and Civil Veterinary Hospital from different animals.According to results of global surveillance by districts of Punjab.Soon after the clinical diagnosis the World Health Organization, about 50,000 cases of was made, the urine samples were collected from the human rabies occur each year [1], the majority of them animals suspected to be rabid.Urine samples were in developing countries [2].Rabies causes fatal collected directly in sterile containers while urinating encephalomyelitis.In India rabies is enzootic and is a or with urethral catheterization.Urine samples serious public health and economic problem [3].The obtained from two healthy animals served as negative appearance of specific rabies disease symptoms is controls.Rabies positive brain homogenate was used preceded by prodromal period in which there are a as positive control.number of non-specific symptoms of malaise [4].
Total RNA from urine samples, positive and Differentiation from other neurological diseases may negative controls was extracted using Qiazol (Qiagen, require extensive investigations.Therefore, diagnosis USA) according to the manufacturer's instructions.is often confirmed late in the course of disease or post- The RNA was subjected to cDNA synthesis using a mortem [5].With the advent of molecular approaches, primer RabN1 (30 pmol/µl) and subjected to 65ºC for it is now possible to detect rabies ante-mortem from 10 min and was later snap cooled on ice and briefly range of biological samples e.g.nuchal skin biopsy [6], spun down.cDNA synthesis was done using highsaliva [7], Cerebrospinal fluid (CSF) [8] and urine capacity cDNA reverse transcription kit (Applied samples [9]. Biosystems, USA).Knowing the feasibility of detection of rabies Reverse transcriptase (Applied Biosystems, USA) from soiled urine sample especially in case of mix was prepared and subjected to conditions 25°C for aggressive rabid animals, the present study was 10 min, 37°C for 2 h, 85°C for 5 min and chilling on ice envisaged to evaluate the importance of TaqMan real for 5 min in a thermal cycler (Eppendorf).RNA and time PCR technique for ante mortem diagnosis of cDNA concentration was measured using Nano Drop rabies from urine samples.

Materials and Methods
ng/µl and quality was checked as a ratio of OD 260/280.

Nested RT-PCR assay:
The procedure used for the Biosystems, Foster City, California) Primer and probe nested RT-PCR based on N (Nucleoprotein) gene was concentrations were optimized according to the manuthat used earlier [10][11][12] with minor modifications.
water was added to make a final volume.Amplification For the second round, 5 µl of first round PCR product was carried out at 50°C for 2 min, 95°C for 10 min, was amplified using Rab Nfor and Rab Nrev and followed by 40 cycles in two steps: 95°C for 15 s, 44°C subjected to thermocycling conditions as first round for 1 min.Amplification, data acquisition and analysis except annealing at 55°C and extension for 1 min.The were carried out by using ABI 7500 instrument and amplified PCR products were loaded on agarose gels ABI prism SDS software which determines the cycle along with positive control, negative control and DNA threshold (Ct) that represents the number of cycles in ladder (100 base pair plus, Fermentas).The agarose which the fluorescence intensity is significantly arose gels were visualized under Geldoc (Bio-Rad).
above the background fluorescence.TaqMan real time PCR assay: Considering the N gene Since, FAT is recommended worldwide as a gold that is most conserved in Lyssavirus and sequence data standard for diagnosis of rabies on neural tissue, after concerned with gene are most exhaustive [7].All death of animal by World Health Organization [13].So, TaqMan primers and probes (Table -2 ) were newly results obtained in TaqMan real time PCR on urine designed at School of Animal Biotechnology, GADVASU samples were compared with FAT for detecting the by the Primer Express 3.0 computer program (Applied sensitivity of this molecular technique.

Table - 1
. Primers used for Nested RT-PCR

Table - 2
. Details of primers and probe for TaqMan real time assay

Table - 3
[14][11][12] of TaqMan real time PCR and Nested RT-PCR with FATResultsvirus shedding, the timing of sample collection, and the type of specimens collected.Moreover, the extent the Amplification with primers Rab N1 and Rab N5 clinical type of rabies (particularly paralytic rabies and yielded 1477 bp first round product.Nested pair of cases with atypical features) influences the outcome of primers (Rab Nfor and Rab Nrev) used for amplilaboratory results[23].Thus, it was concluded that fication in second round yielded 762 bp product as TaqMan real time PCR could be a feasible approach as reported[10][11][12].By nested RT-PCR, viral RNA could compared to Nested RT-PCR for ante mortem diagnosis be diagnosed in 6/21 (28.57%) in urine samples (Table-of rabies.This study highlights its utility in establishing 3) with a sensitivity of 62.50%.antemortemdiagnosis of rabies using urine samples In TaqMan real time PCR samples in which within a few hours.thresholdcyclenumber (Ct) values were found to be in Conclusion the range of 20-35 were considered positive and above 35 were considered negative[14].With this technique TaqMan real time PCR can serve as more sensitive viral RNA could be diagnosed in 11/21 (52.38%) urine and viable approach for the intravitam diagnosis of samples sensitivity of 78.94% was obtained when rabies as compared to Nested RT-PCR for detection of compared with gold standard immunofluorescence test rabies from urine of suspected animals.(FAT) on brain (Table-3).Percent of positivity (52.38 NA-Not Available, + Positive, -Negative