Evaluation of Nucleic Acid Sequence Based Amplification (NASBA) and Reverse Transcription Polymerase Chain Reaction for Detection of Coxsackievirus B3 in Cell Culture and Animal Tissue Samples

Document Type : Research Paper


1 Department of Biotechnology, Maleke Ashtar University, P.O. Box 15875-1774, Tehran, I.R. Iran

2 Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran

3 Neuroscience Research Center, Shahid Beheshti Medical University, P.O. Box 16765-3718, Tehran, I.R. Iran


Enteroviruses are the causative agents of a number of diseases in humans. Group B coxsackieviruses are believed to be the most common viral agents responsible for human heart disease. Genomic data of enteroviruses has allowed developing new molecular approaches such as Nucleic Acid Sequence Based Amplification (NASBA) for detection of such viruses. In this study, coxsackievirus B3 (CVB3) was detected in virus-infected cell culture and specimens of artificially infected mice with specific primers using Reverse Transcription - Polymerase Chain Reaction (RT-PCR) and NASBA techniques. According to the results, both techniques could be used for the detection of viruses in cell culture and artificially infected animals. NASBA reaction was simpler to perform than RT-PCR. The only variable factor that had to be optimized with NASBA is KCl concentration. The optimal concentration of KCl was determined as 90 mM. Serial dilutions of 1 mg of total RNA showed that both RT-PCR and NASBA could detect the virus at 10-5 dilution. Analyses of heart and spleen samples from infected animals were positive for presence of Coxsackievirus B3 with both RT-PCR and NASBA. In conclusion, NASBA offers some advantages over RT-PCR and is a suitable alternative technique for the sensitive detection of CVB3 in contaminated samples.



Human enteroviruses are causative agents of a number of diseases and are widespread throughout the world. Enteroviruses, particularly the group B coxsackieviruses, are believed to be responsible for the most common viral human myocarditis (Woodruff, 1980). It is speculated that they may play a role in development of idiopathic dilated cardiomyopathy (IDC) (Lerner and Wilson, 1987). Enteroviruses have a strong tropism for the myocardium (Lerner and Wilson, 1987). Inoculation of coxsackieviruses in experimental animals induces myocarditis, which can evolve into cardiomyopathy with associated virus persistence for several weeks after primary infection (Kandolf et al., 1987; Kawai et al., 1978). Evidence for involvement of enteroviruses in acute myocarditis was suggested by detection of neutralizing IgM and IgG antibodies specific to the group B coxsackieviruses (Baboonian et al., 1997). The presence of coxsackievirus B3 (CVB3) specific IgM in patient’s sera after a phase of myocarditis indicated that enteroviruses could also establish chronic infection, which would result in IDC (Muir and Archard, 1994). At this stage, the detection of enteroviral particles or viral antigens in the myocardium by using conventional biological techniques was unsuccessful (Bowles and Towbin, 2000; Woodruff, 1980).
Genomic data of enteroviruses has allowed the development of new molecular approaches for detection of viruses (Rotbart, 1990). Hybridization studies, that use slot-blot or in situ hybridization, have demonstrated positive enterovirus detection in the myocardium of patients with acute myocarditis and IDC (Kandolf et al., 1987). The use of RT-PCR has allowed more specific and sensitive enterovirus detection in the human myocardium (Rey et al., 2001; Schwaiger et al., 1993; Severini et al., 1993; Grasso et al., 1992; Rotbart, 1990). Enteroviral RNA has been observed in heart tissue from approximately 25% of patients suffering from idiopathic acute myocarditis but also in 25% of patients undergoing transplantation due to end-stage chronic myocarditis. These findings suggest that molecular techniques could be valuable tools for clinical diagnosis of enterovirus endomyocardial infection (Baboonian et al., 1997).
     Nucleic acid amplification techniques, such as PCR, are highly sensitive and specific tools for the amplification and detection of pathogen-specific sequences. As an alternative to PCR, the nucleic acid sequence-based amplification (NASBA) technique has been developed for detecting specific nucleic acids (Compton, 1991). NASBA is an isothermal nucleic acid amplification reaction, using two specific oligonucleotide primers (one containing a T7 promoter sequence) and three enzymes, AMV reverse transcriptase, RNase H and T7 RNA polymerase (Fig. 1) (Deiman et al., 2002; Jean et al., 2001; Compton, 1991; Kievits et al., 1991). NASBA can amplify DNA and RNA template sequences and its major reaction product is RNA complementary to the target sequence (Rodríguez-Làzaro et al., 2004). NASBA has proven to be a useful technique for the highly sensitive detection of viral nucleic acids in clinical samples (Loens et al., 2006; Yoo et al., 2005; Chan and Fox, 1999), microbial pathogens in food and environmental samples (Fykse et al., 2007; Jean et al., 2004; Cook, 2003). With some modifications, It has also been used in the quantization of viral nucleic acids (Schneider et al., 2005; Leone et al., 1998).
    In the present study, NASBA and RT-PCR techniques were developed and evaluated for diagnosis of CVB3 in cell culture and artificially infected animals.


Virus culture and RNA extraction: CVB3 Nancy strain, ATCC number VR-30 was grown on Vero cell culture and used as a positive control. Virus-infected cultures were collected 48 h after infection when all the cells showed the characteristic cytopathic effect. Uninfected cell culture was also used as a negative control. Aliquots of positive and negative cultures were collected and stored at -80ºC for the purpose of RNA extraction. Specimens were frozen and thawed three times and clarified by centrifugation at         13,000 ×g for 10 min at 4ºC. Viral nucleic acid was extracted from the supernatant by the TriZol RNA extraction kit (Fermentas, Germany), according to the manufacturer’s instructions.

Artificially infected animals: Eight Balb/c mice, aged 8 weeks, were divided into 2 same groups. Group 1, the control group, inoculated with medium, while group 2 inoculated with the cultured virus. A sample containing 105 plaque forming units (pfu) of the CVB3 was used to inoculate Balb/c mice via the intraperitoneal route (IP). After a week, a booster dose was used to inoculate again and 2 weeks later, samples were collected from heart, blood, feces and spleen, and then stored at -80ºC for RT-PCR and NASBA analyses.

Primer design: Primers were designed by the Oligo 6 software, (Molecular Biology Insights, Inc., Cascade, CO), according to the CVB3 genomic sequence in GenBank (Accession No: FJ000001). The P1 and P2 primers for the RT-PCR were designed on the basis of the VP1 and VP3 genes to amplify a 710 bp segment of CVB3 RNA. The NASBA primers, designated P3 and P4, were also directed against the VP3 and VP2 genes of CVB3, respectively. The P3 primer contains the T7 RNA polymerase promoter which is recognized by T7 RNA polymerase during the amplification step leading to the production of single stranded anti-sense RNA. Because the amplification of longer products is less efficient when using the NASBA reaction, the primers were selected in a manner to amplify a 225 bp product. The primers were checked for secondary structures and dimmers and primers’ specificity were determined by the Blastn program. The primers were designed in a manner to specifically detect CVB3 (Table 1).

Reverse Transcription- Polymerase Chain Reaction (RT-PCR): RNA was amplified using a two step RT-PCR method. For reverse transcription, samples were denatured for 10 min at 65ºC followed by incubation at 37ºC during which reverse transcriptase was added. After incubation for 60 min, the reaction was stopped by heating at 95ºC for 5 min. PCR reaction was carried out followed by 35 cycles of at 94ºC for 45 s, 55ºC for 45 s, and 72ºC for 30 s. PCR products was analyzed by 1% agarose gel electrophoresis. RT-PCR products were confirmed by digestion with appropriate restriction enzymes.

Nucleic Acid Sequence Based Amplification (NASBA): The NASBA was carried out as described by Kievits et al. (1991) with some modifications.  The reaction mixture was prepared in a total volume of     20 ml containing  5 ml of extracted RNA, 40 mM Tris (pH 8.5), 12 mM MgCl2, 70 mM KCl, 5 mM dithiotritol (DTT), 1 mM of each deoxyribonucleoside triphosphate (dNTP), 2 mM each ribonucleoside triphosphate, 15% (v/v) dimethylsulphoxide (DMSO), and 5 pmol of each primer. The reaction mixture was incubated at 65ºC for 5 min and subsequently transferred to 41ºC. After 5 min, an enzyme mixture containing      2.1 mg of BSA, 0.08 U of RNase H (0.08 U/ml) of RNase H, 32 U of T7 RNA polymerase (32 U/ml) and 6.4 U of Avian Myeloblastosis Virus - Reverse Transcriptase (AMV-RT) (6.4 U/ml) was added to the mixture and the resulting reactions were performed at 41ºC for 90 min. NASBA products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide staining.


In this study, the RT-PCR and NASBA assays were performed for the detection of CVB3 in virus-infected cultures and specimens of artificially infected mice. The results of NASBA were compared with those of RT-PCR.

Virus detection in cell culture: RNA was extracted from CVB3 infected cells and used to determine the appropriate reaction conditions and evaluate the sensitivities of the NASBA and RT-PCR assays. The size of NASBA and RT-PCR products were 225 and 710 bps, respectively. The NASBA and RT-PCR gave positive specific amplification signals when challenged with the RNA of CVB3 infected cells.
     The NASBA assay of CVB3, was optimized for KCl concentration. The level of amplified product increased as the concentration of KCl was increased from 50 to 90 mM (Fig. 2). The sensitivity of NASBA and RT-PCR were determined by testing serial log10 dilutions of RNA obtained from CVB3-infected cells. The results showed that both NASBA and RT-PCR could detect CVB3 at the 10-5 dilution of a 1 μg RNA sample extracted from the virus-infected cells (Fig. 3).
Assessment of specificity: Poliovirus vaccine was used for assessment of both NASBA and RT-PCR specificities. Neither NASBA nor RT-PCR could detect poliovirus extracted RNA under the conditions described for CVB3.

Analysis of artificially infected animals: The NASBA and RT-PCR were further evaluated by testing infected mice. Balb/C mice were inoculated by the IP route with 105 PFU of the virus. Samples were collected 2 weeks post-infection from heart, spleen, blood, and feces. The results of RT-PCR on heart and spleen samples were positive, but not on blood and feces samples. The NASBA also detected the virus in heart and spleen specimens (Fig. 4).


Enteroviruses are responsible for several diseases in humans. Among enteroviruses, CVB3 is the most important viral agent of myocarditis and IDC (Lerner and Wilson, 1987; Woodruff, 1980). Detection of enteroviral RNA in myocardial tissue of patients with IDC has only been demonstrated using slot blot (Bowles, et al., 1986), in situ hybridization (Kandolf, 1988), and PCR (Shigekazu, et al., 2000; Jin, et al., 1990). Detection of enteroviral RNA in some diseases was demonstrated using the NASBA technique (Capaul and Gorgievski-Hrisoho, 2005; Ginocchio et al., 2005; Heim and Schumann, 2002; Fox et al., 2002).
     In the present study, NASBA and RT-PCR techniques were used to detect CVB3 in cell culture and animal samples. The results revealed that RT-PCR and NASBA are suitable for detection of the virus in cell culture. The sensitivity of these techniques was determined by serial dilution of total RNA. Both the techniques could detect viral RNA at the 10-5 dilution of a 1 mg RNA sample derived from CVB3-infected Vero cells (Fig. 3). Experiments on animal models revealed that NASBA is a suitable alternative to RT-PCR for sensitive detection of the virus in heart and spleen samples. But there were no positive amplification signals in the case of feces and blood samples by both RT-PCR and NASBA. It may be due to the presence of inhibitors such as complex polysaccharides in feces which prevent amplification during RT-PCR and NASBA procedures. The negative results with blood samples may be related to low levels of the virus. The specificity test was only examined with the poliovirus. Of course, it is not enough for confirming the specificity of the techniques, but there was limited access to all entroviruses and the results showed an absence of amplification by both RT-PCR and NASBA techniques.
 In a previous study, small amounts of viral genomes were detected in a latent state with focal distribution in the myocytes (Kandolf, 1988). Grasso et al. (1992) detected CVB3 at the 10-5-10-6 dilutions of a 1 mg RNA sample derived from coxsackievirus B3-infected cells. Other studies have shown that NASBA and RT-PCR produce comparable results and are significantly more sensitive than the virus culture technique (Houde et al., 2006; Loens et al., 2006). Rutjes et al. (2005) have found that the rapid real-time NASBA assay is slightly less sensitive than the RT-PCR for the detection of entrovirus in water.
    According to the present study, both NASBA and RT-PCR have the same sensitivity and specificity for detection of CVB3. NASBA offers some advantages over RT-PCR because it is an isothermal reaction, obviating the need for a thermal cycler and the optimal annealing temperature for primers does not have to be determined empirically. NASBA is a single step reaction, which decreases the risk of cross contamination in comparison to the two step RT-PCR. It is important to detect CVB3 in clinical laboratories in order to avoid false positive results. Because NASBA is performed in a single, closed tube format, it minimizes the risk of amplicon contamination due to fewer handling step. The amplification conditions for NASBA are generally constant, and optimization of conditions for each new assay can be simpler than RT-PCR. The concentrations of enzymes and primers used for NASBA are standardized and do not differ from assay to assay. The only variable factor that has to be optimized in NASBA is the KCl concentration. It is easily performed in a single experiment using a KCl concentration range of 50-120 mM. However, most targets will have optimal KCl concentrations between 70 and 90 mM. In this study, the optimal concentration of KCl was determined as 90 mM. NASBA is easier to perform and produces results more rapidly than RT-PCR.
In conclusion, NASBA is simpler and safer to perform than RT-PCR for detection of coxsackievirus B3. There is no advantage in sensitivity and specificity between the two techniques. Therefore, we recommend the NASBA as an alternative method for detection of this virus in cell culture.  Performance of NASBA on samples obtained from cell cultures as well as tissue biopsies from infected animals is promising. However, its efficacy in detecting the CB3 virus in human samples awaits further investigations.

Baboonian C, Davies MJ, Booth JC, Mc Kenna WJ (1997). Coxsackie B viruses and human heart disease. In: The coxsackie B viruses. Berlin-Heidelberg, Springer Verlag. pp. 31-52.
Bowles NE, Archard LC, Olsen EGJ, Richardson PJ (1986). Detection of Coxsackie-B-virus-specific RNA sequences in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy. Lancet 1: 1120-1123.
Bowles NE, Towbin JA (2000). Molecular aspects of myocarditis. Curr Infec Dise Repo. 2: 308-314.
Capaul SE, Gorgievski-Hrisoho M (2005). Detection of enterovirus RNA in cerebrospinal fluid (CSF) using NucliSens EasyQ Enterovirus assay. J Clin Virol. 32: 236-240.
Chan AB, Fox JD (1999). NASBA and other transcription-based amplification methods for research and diagnostic microbiology. Rev Med Microbiol. 10: 185-196.
Compton J (1991). Nucleic acid sequence-based amplification. Nature 350: 91-92.
Cook N (2003). The use of NASBA for detection of microbial pathogens in food and environmental samples. J Microbiol Meth. 52: 165-174.
Deiman B, van Aarle P, Sillekens P (2002). Characteristics and applications of nucleic acid sequence-baced amplification (NASBA). Mol Biotechnol. 20: 163-179.
Fox JD, Han S, Samuelson A, Zhang Y, Neale ML, Westmoreland D (2002). Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the nucli-sens® Basic kit. J Clin Virol. 24: 117-130.
Fykse EM, Skogan G, Davies W, Olsen JS, Blatny JM (2007). Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification. Appl Environ Microbiol. 73: 1457-1466.
Ginocchio CC, Zhang F, Malhotra A, Manji R, Sillekens P, Foolen H, Overdyk M, Peeters M (2005). Development, technical performance, and clinical evaluation of a NucliSens basic kit application for detection of enterovirus RNA in cerebrospinal fluid. J Clin Microbiol. 43: 2616-2623.
Grasso M, Arbustini E, Silini E, Diegoli M, Percivalle E, Ratti G, Bramerio M, Gavazzi A, Vigano M, Milanesi G (1992). Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction. Am J Cardiol. 69: 658-664.
Heim A, Schumann J (2002). Development and evaluation of a nucleic acid sequence-based amplification (NASBA) protocol for the detection of enterovirus RNA in corebrospiral fluid samples. J Virol Meth. 103: 101-107.
Houde A, Leblanc D, Poitras E, Ward P, Brassard J, Simard C, Trottier YL (2006). Comparative evaluation of RT-PCR, nucleic acid sequence-based amplification (NASBA) and real-time RT-PCR for detection of noroviruses in faecal material. J Virol Methods. 135: 163-172.
Jean J, Blais B, Darveau A, Fliss I (2001). Detection of hepatitis A virus by the nucleic acid sequence- based amplification technique and comparison with reverse transcription-PCR. Appl Envir Microbiol. 67: 5593-5600.
Jean J, D’Souza DH, Jaykus LA (2004). Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods. Appl Environ Microbiol. 70: 6603-6610.
Jin O, Sole MJ, Butany JW, Chia WK, McLaughlin PR, Liu P, Liew CC (1990). Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction. Circulation  82: 8-16.
Kandolf R (1988). the impact of recombinant DNA technology on the study of enterovirus heart disease. In: Coxsackievirus: A General Update. New York, Plenum Publishing, pp. 303-305.
Kandolf R, Ameis D, Kirschner P, Canu A, Hofschneider PH (1987). In situ detection of entroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease. Proc Natl Acad Sci USA. 84: 6272-6276.
Kawai C, Matsumori A, Kitawa Y, Takatsu T (1978). Virus and the heart: viral myocarditis and cardiomyopathy. Prog Cardiol. 7: 141-162.
Kievits Tim, Gemen Van B, Van Strijp D, Schukkink R, Dirks M, Adriaanse H, Marek L, Sooknanan R, Lens P (1991). NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. J virol Meth. 35: 273-286. 
Leone G, Van Schijndel H, Van Gemen B, Kramer FR, Schoen CD (1998). Molecular beacon probes combined with amplification by NASBA enable homogeneous, real time detection of RNA. Nuc A Res. 26: 2150-2155.
Loens K, Beck T, Goossens H, Ursi D, Overdijk M, Sillekens P, Ieven M (2006). Development of conventional and real-time nucleic acid sequence-based amplification assays for detection of Chlamydophila pneumoniae in respiratory specimens. J Clin Microbiol. 44: 1241-1244.
Loens K, Goossens H, de Laat C, Foolen H, Oudshoorn P, Pattyn S, Sillekens P, Ieven M (2006). Detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-PCR, in children with acute respiratory infections during a winter season. J Clin Microbiol. 44: 166-171.
Lerner AM, Wilson AM (1987). Virus myocardiopathy. Prog Med Virol. 15: 63-91.
Muir P, Archard LC (1994). There is evidence for persistent enterovirus infections in chronic medical conditions in humans. Rev Med Virol. 4: 245-250.
Rey L, Lambert V, Wattre P, Andreoletti L (2001). Detection of enteroviruses ribonucleic acid sequences in endomyocardial tissue from adult patients with chronic dilated cardiomyopathy by a rapid RT-PCR and hybridization assay. J Med Virol. 64: 133-140.
Rodríguez-Làzaro D, Lloyd J, Ikonomopoulos J, Pla M, Cook N (2004). Unexpected detection of DNA by nucleic acid sequence-based amplification technique. Mol Cell Probes. 18: 251-253.
Rotbart HA, (1990). Nucleic acid detection systems for enteroviruses. Clinic Microbiol Rev. 4: 156-168.
Rutjes SA, Italiaander R, van den Berg HH, Lodder WJ, de Roda Husman AM (2005). Isolation and detection of enterovirus RNA from large-volume water samples by using the NucliSens miniMAG system and real-time nucleic acid sequence-based amplification. Appl Environ Microbiol. 71: 3734-3740.
Schneider P, Wolters L, Schoone G, Schallig H, Sillekens P, Hermsen R, Sauerwein R (2005). Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum. J Clin Microbiol. 43: 402-405.
Schwaiger A, Umlauft F, Weyrer K, Larcher C, Lyon J, Mühlberger H, Dietze O, Grnewald K (1993). Detection of enteroviral ribonucleic acid in myocardial biopsies from patients with idiopathic dilated cardiomyopathy by polymerase chain reaction. Am Heart J. 126: 406-410.
Severini GM, Mestrioani L, Falashi A, Camerini F, Giacca M (1993). Nested polymerase chain reaction for high-sensitivity: detection of enteroviral RNA in biological samples. J Clin Microbiol. 31: 1345-1349.
Shigekazu F, Kitaura Y, Ukimura A (2000). Evaluation of Viral Infection in the Myocardium of Patients with Idiopathic Dilated Cardiomyopathy. J Am College Cardiol. 36: 1920-6.
Woodruff JF (1980). Viral myocarditis. Am J Pathol. 101: 427-479.
Yoo JH, Choi JH, Choi SM, Lee DG, Shin WS, Min WS, Kim CC (2005). Application of nucleic acid sequence-based amplification for diagnosis of and monitoring the clinical course of invasive aspergillosis in patients with hematologic diseases. Clin Infect Dis. 40: 392-398.