Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 2, 2011, pp. 110-117

Indian Journal of Medical Microbiology, Vol. 29, No. 2, April-June, 2011, pp. 110-117

Original Article

Development of a new method for diagnosis of Group B Coxsackie genome by reverse transcription loop-mediated isothermal amplification

K Jaianand¹, N Saravanan², P Gunasekaran¹, AK Sheriff¹

¹ Department of Virology, King Institute of Preventive Medicine, Guindy, Chennai- 600 032, India
² Department of Biotechnology, Adhiyamaan College of Engineering, Hosur - 635 109, India

Correspondence Address: K Jaianand Department of Virology, King Institute of Preventive Medicine, Guindy, Chennai- 600 032 India jaianand.k@lifecellinternational.com

Date of Submission: 30-Sep-2009
Date of Acceptance: 23-Apr-2010

Code Number: mb11028

PMID: 21654103

DOI: 10.4103/0255-0857.81780

Abstract

Keywords: Coxsackie B viruses, mediated isothermal amplification, non-polio enterovirus, real-time polymerase chain reaction, reverse transcription loop-mediated isothermal amplification

Introduction

Enteroviruses belong to the Picornaviridae family of viruses and are further organised into the subgenera polioviruses, Coxsackieviruses (groups A and B), and echoviruses. More than 60 serotypes have been identified; but only the first 61 have been classified; 3 serotypes comprise the polioviruses, 23 serotypes comprise Coxsackievirus group A, 6 serotypes comprise Coxsackievirus group B, and 29 serotypes comprise the echoviruses. The Coxsackieviruses are icosahedral nonenveloped viruses that are approximately 30 nm in diameter. The genome is made of a single-stranded linear molecule of RNA. Coxsackievirus infection is the most common cause of viral heart disease. Group B viruses cause spastic paralysis; other diseases associated with group B Coxsackievirus infections include herpangina, pleurodynia, myocarditis, pericarditis, meningoencephalitis, aseptic meningitis and colds.

The routine diagnostic methods for Coxsackievirus by virus cell culture are followed by serum neutralisation test, the gold standard for enterovirus typing. This method is generally reliable, but also labour-intensive, time-consuming, and costly. Furthermore, the supply of antisera is limited and the problem of "untypeable" enteroviruses is frequently encountered. The comparatively slow procedures of in vivo amplification of Coxsackieviruses in the cell culture may be replaced by rapid in vitro amplification of viral RNA sequences by the loop-mediated isothermal amplification (LAMP).

More recently, several investigators have reported on fully automatic Real-Time Polymerase Chain Reaction (RT- PCR) assays for the detection of enteroviruses. Moreover, PCR-based technology holds more promise. As yet, however, PCR has not established a routine foothold in clinical laboratories because it is a time-consuming complex and needs a high-precision thermal cycler. Reaction equipment that is much simpler and amenable for use in hospital laboratories is required. By contrast, the LAMP assay reported here is advantageous owing to its simple operation, rapid reaction, and easy detection. LAMP operates under isothermal conditions at 63°C for 1 hour. Therefore, no time is lost as a result of changes in temperature, as is the case with thermal cycling with PCR. Moreover, LAMP requires only simple reaction equipment; it can be performed using a regular laboratory bath or heat block that provides a constant temperature of 63°C. It is even possible to determine the reaction directly with the naked eye, without electrophoretic analysis, unlike PCR. Since the LAMP assay is simple and relatively easy to perform, even a clinical microbiological laboratory that has not performed PCR or molecular testing could introduce this technology.

Materials and Methods

Clinical samples

A total of 150 CSF and stool samples [Table - 1] received from patients with a clinical diagnosis of meningitis, acute flaccid paralysis were used for evaluation in this study. The samples were collected during the period between days 1 and 7 after the onset of symptoms. All the samples were stored at −80°C until further investigation. In addition, a panel of five stool samples and four CSF samples collected from healthy individuals was also included as negative controls. Prior to reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay, out of these 150 samples, 31 samples were investigated for the presence of non-polio enterovirus/Coxsackie B specific RNA by RT-PCR and RT-LAMP.

Virus strains

Prototype strains of Coxsackie B1-B5 viruses (CVB 1-Conn-5, CVB 2-Ohio1, CVB 3-Nancy, CVB 4-JVB, CVB 5-Faulkner) were propagated in HEp-2 cell line. After complete cytopathic effect (CPE), the culture was harvested after three cycles of freezing and thawing. Initial clarification was carried out by centrifugation at 1000 ×g for 15 min at 4°C and supernatant was stored at −80°C.

Inoculation of processed samples

The culture tubes were examined under a microscope to ensure that the cells were healthy, confluent and free from microbial contamination. Then, the growth medium was decanted and replaced with 1 ml of 2% Eagle′s Minimal Essential medium (MEM; PAN Biotech, Aidenbach, Germany). Two culture tubes of each cell type were inoculated with 0.2 ml of specimen extract. Uninoculated tubes of each cell type served as controls. The tubes were incubated at 36°C in the stationary-sloped position at 5° inclination.

Cell culture

HEp-2 cell line, RD cells derived from human rhabdomyosarcoma, and L20B-genetically engineered mouse cell line expressing the human poliovirus receptor were maintained at 37°C under 5% CO ₂ by regular subculturing at periodic intervals for 4-5 days in Eagle′s MEM (PAN Biotech). The cultures were examined daily using an inverted microscope for the appearance of CPE for at least 5 days, and the daily observation are recorded. Control cultures should maintain their normal healthy appearance [Figure - 1]. Tubes showing rounding and detachment of cells from the surface is the characteristic CEF of enterovirus. When the CPE was greater than 75% [Figure - 2], the culture was collected and stored frozen at −70°C.

Identification of enteroviruses from cell culture

If the specimen showed no CPE in the both cell lines at the end of the second passage, it was reported as negative. If the specimen was positive in L20B and RD cell lines, poliovirus was identified only from L20B harvest. If the specimen was positive in RD cell line and negative in L20B, then the RD harvest was passed into L20B cell line, and if CPE was not observed in RD/L20B, it was reported as non-polio enterovirus/Coxsackie B viruses. If the CPE was observed in L20B from RD harvest, the poliovirus was identified from RD/L20B harvest.

Identification of enteroviruses isolates by neutralisation test

The standard procedure for virus isolate identification was performed using micro technique. The microtitre plate was labelled with sample number, date, and dilution of sample to be used. 50 μl of each of the antiserum pools was added to rows A-D: first well for polio pool, second well for Coxsackie pool and third to ninth wells for echo pools. 50 μl of MEM (PAN Biotech) was added to virus control wells, i.e. rows E-H and to column 10. 100 μl of MEM was added to the cell control wells G11-H12. The test sample was diluted serially from 10⁻¹ to 10⁻⁷ and 50 μl of virus sample was added to the test wells that contained 100 TCID ₅₀ /50 μl from 10⁻³ and 10⁻⁴ dilution.

Confirmation of mixture of polio and non-polio enterovirus by neutralisation test

A combination of polio and non-polio enterovirus was isolated in RD cell line. Confirmation of the isolates was done from the pool of non polio enterovirus virus (NPEV). 10⁻¹ dilution was prepared and Coxsackievirus neutralisation test was done as described to confirm the group B Coxsackievirus identity. Calculation of virus titre was done by Karber′s formula.

RNA extraction

RNA extraction was carried out using Qiagen Extraction kit (QIAGEN, Hilden, Germany) according to the manufacture′s instruction from the tissue culture fluid of clinical isolates and reference strains. Nucleic acids were dissolved in 30 μl of distilled diethylpyrocarbonate (DEPC) (Sigma, St. Louis, MO, USA) treated water. All RNA samples were stored at −80°C.

Reverse transcription and PCR

cDNA was synthesised in a 20 μl reaction mixture containing 75 mM Tris-HCl (pH 8.3) (Sigma), 3 mM MgCl ₂ (Sigma), 10 mM dTT (Roche Diagnostic, Penzberg, Germany), 0.2 mM each of dNTPs (Roche Diagnostic), 50 pmol of primer "B" 5′-ATTGTCACCATA AGCAGCCA-3′ (Roche Diagnostic) positions 580-599 (refer to the Coxsackie B-1 sequence), 10 U of avian myoblastosis virus reverse transcriptase (Roche Diagnostic), and 5 μl of RNA isolated from clinical samples and the reference viruses. After incubation at 37°C for 60 min, 1 μl of cDNA was added to PCR master mix containing 50 mM KCl (Sigma), 10 mM Tris-HCl (pH 8.9) (Sigma), 3.6 mM MgCl ₂ (Sigma), 10 mM dTT, 0.2 mM each of dNTPs (Roche Disgnostic, US A), 50 pmol of each primer B and primer "A1" 5′-CAAGCACTTCTGTTTCCCCGG-3′ (Roche Diagnostic) positions 160-180 and 2.5 U of Taq DNA polymerase (Roche Diagnostic).

Sensitivity of RT-PCR for the detection of Coxsackie B by gel analysis

The electrophoresis of amplified PCR product was carried out in 1.5% low melting agarose gel (Sigma). Desired amplified product was separated and the sensitivity of RT-PCR for the detection of the Coxsackie B 5′ UTR gene was observed by agarose gel analysis with a detection limit of 200 copy numbers.

Loop-mediated isothermal amplification

The oligonucleotide primers used for RT-LAMP amplification of Coxsackieviruses were designed from the 5′ UTR portion, to identify the conserved regions using DNASIS software. The potential target region was selected from the aligned sequences, and RT-LAMP primers were designed. A set of six primers comprising two outer, two inner, and two loop primers that recognise eight distinct regions on the target sequence was designed by employing the LAMP primer-designing support software program (Primer Explorer 4, Eiken, Japan). The primers were selected based on the criteria described previously. ^[1]

Primer design for LAMP

The two outer primers were described as being forward outer primer (F3) and backward outer primer (B3). The inner primers were described as being Forward Inner Primer (FIP) and Backward Inner Primer (BIP). Furthermore, two loop primers, viz., Forward Loop Primer (FLP) and Backward Loop Primer (BLP), were designed to accelerate the amplification reaction. FIP consists of a complementary sequence of F1 and a sense sequence of F2. BIP consists of a complementary sequence of B1 and a sense sequence of B2. FIP and BIP were high-performance liquid chromatography purified primers. The FLP and BLP primers were composed of the sequences that are complementary to the sequence between the F1 and F2 and B1 and B2 regions, respectively. The details of the each primer with regard to their positions in the genomic sequences are shown in [Figure - 3].

Viral RNA extraction

The genomic viral RNA was extracted from 100 μl of standard viral samples by using the QIAamp Viral RNA Mini kit (QIAGEN) according to the manufacturer′s protocol. The RNA was eluted from the QIAspin columns in a final volume of 80 μl of elution buffer and was stored at −70°C until use.

Strategies of the amplification reaction

The RT-LAMP reaction was carried out in a total 25 μl reaction volume, using the Loopamp RNA amplification kit (Invitrogen, CA, USA) containing 50 pmol each of the primers FIP and BIP (Sigma), 5 pmol each of the outer primers F3 and B3 (Sigma), 1.4 mM deoxynucleoside triphosphates (Fermentas, Burlington, Canada), 0.8 M betaine (Sigma), 0.1% Tween 20 (Sigma), 10 mM (NH ₄ ) ₂ SO ₄ (Sigma), 8 mM MgSO ₄ (Sigma), 10 mM KCl (Sigma), 20 mM Tris-HCl (pH 8.8) (Sigma), 8 units of Bst DNA polymerase (New England Biolabs, Ipswich, MA, USA), 0.625 units of AMV reverse transcriptase (Invitrogen, CA, USA), and 2 μl of RNA template. Positive and negative controls were included in each run, and all precautions to prevent cross-contamination were observed.

Analysis of RT-LAMP product

Following incubation at 63°C for 60 min, 10 μl aliquots of RT-LAMP products were electrophoresed on a 1.5% agarose gel in Tris-borate buffer (Sigma) followed by staining with ethidium bromide (Sigma) along with 100 bp DNA ladder (Fermentas) and visualisation on a UV transilluminator at 302 nm [Figure - 4].

Visualisation by the naked eye

In order to facilitate the field application of the RT-LAMP assay, the monitoring of RT-LAMP amplification was also carried out with inspection by the naked eye. Following amplification, the tubes were inspected for white turbidity using the naked eye after a pulse spin to deposit the precipitate in the bottom of the tube. The inspection for amplification was also performed through observations of colour change following the addition of 1 μl of SYBR Green I dye (Invitrogen, CA, USA) to the tube. In the case of positive amplification, the original orange colour of the dye would change into green that can be judged under natural light as well as under UV light (302 nm) with the help of a hand-held UV torch lamp. In case there is no amplification, the original orange colour of the dye would be retained. This change of colour is permanent [Figure - 5] and thus can be kept for record purposes.

Results

A total of 150 CSF and stool samples [Table - 1] were inoculated in RD and L20B cell lines, and the outcome of the virus isolation tests in 31 samples′ viral CPE was obtained in RD cells either post-inoculation or post-passage and no CPE was obtained when the RD isolate was passed into L20B cells, which are likely to be NPEV. In 81 samples, no viral CPE was observed in post-inoculation or post-passage of L20B and RD cell cultures for any of the inoculated cultures or specimens of the cases, which are concluded to be negative. Seven samples were L20B positive and were identified as being NPEV positive.

A one-step, single-tube, accelerated, quantitative RT-LAMP assay was standardised for the rapid detection of group B Coxsackieviruses by targeting the highly conserved regions of the 5′ -UTR gene based on multiple sequence alignments of all the circulating strains. The details of the each primer with regard to their positions in the genomic sequences are shown in [Table - 2]. The detection of gene amplification is accomplished by real-time monitoring of turbidity at 63°C. The result indicated that the minimum time required for the initiation of amplification was 10 min with viral RNA preparations. It was also observed that there is continuous amplification of the target sequence as revealed through increased turbidity compared to the negative control having no template, wherein the turbidity got fixed around 0.01, well below the threshold value. None of the positive samples tested over multiple times showed positivity in terms of increased turbidity after 45 min. Therefore, a sample with a Tp value of 45 min and turbidity above the threshold value of 0.1 was considered positive.

Evaluation and comparison of RT-LAMP and RT-PCR for detection of Coxsackieviruses in clinical samples

The applicability of the RT-LAMP assay for the clinical diagnosis of Coxsackieviruses was validated with CSF and stool samples. The results were compared with those from RT-PCR. A total of 40 samples comprising 31 positive samples and 9 negative samples were used in this study for comparative evaluation. None of the RT-PCR-positive samples were missed by RT-LAMP, thereby indicating a higher sensitivity of the RT-LAMP assay. All nine healthy serum samples were also negative by both the tests, thereby ruling out the possibility of false positivity and thus establishing the specificity of the selected primer sets for the group B Coxsackie RT-LAMP assay. The RT-LAMP assay also picked up more positive samples than RT-PCR, virus isolation, and neutralisation test [Figure - 4]. The field applicability of the RT-LAMP assay was also validated by employing a SYBR Green I-mediated naked-eye visualisation test. Following incubation at 63°C for 30 min in a water bath, the monitoring of RT-LAMP amplification was accomplished through visualisation by the naked eye with the addition of 1 μl of SYBR Green I (1:1000) dye to the amplified products [Figure - 5]. The comparative evaluation of this field-based, SYBR Green I-based RT-LAMP assay with 10 clinical samples randomly selected from the above-described 31 samples revealed a very good concordance of 90% with RT-PCR [Figure - 6].

Discussion

Group B Coxsackievirus consist of six serotypes (1- 6), classified within the enterovirus genus of the family Picornaviridae. Coxsackie B viruses are the aetiological agents of a wide spectrum of human diseases, including mild respiratory infection, aseptic meningitis, and fatal myocarditis. Outbreaks of Coxsackie B viruses′ infection occur annually throughout the world. ^[2] Infection of newborns and infants by these viruses can induce paralysis, aseptic meningitis, and febrile illnesses that can be fatal, while the infections in adults are mostly asymptomatic. ^[3],[4] The results of the serotype-specific descriptive analysis by sex, age group, specimen type and collection month, and outcome indicate similarities and patterns characteristic of individual Coxsackie B viruses.

The major clinical presentations of Coxsackievirus B5 infections are similar to those observed for other group B Coxsackieviruses and include myopericarditis and neonatal systemic illness (encephalomyocarditis syndrome), aseptic meningitis, meningoencephalitis, and acute flaccid paralysis. Hand, foot, and mouth disease and herpangina also have been reported, and a potential association with development of type 1 diabetes has been suggested. ^[5],[6] Approximately half of all Coxsackievirus B5 detections came from young infants, and CSF was the most common source. The summer-fall seasonality in Coxsackievirus B5 detections was more prominent than for most other serotypes. ^[7]

Clinical illnesses associated with Coxsackievirus B2 include aseptic meningitis, myocarditis, and neonatal systemic illness. ^[7] Outbreaks in settings such as football teams and summer camps were reported. ^[8] The virus has an endemic pattern of circulation with year-to-year variability and has consistently appeared among the top 15 serotypes. The proportion of children aged <1 year among reports of Coxsackievirus B2 detections exceeded 60%, and CSF was the most common source of detection.

Outbreaks of Coxsackievirus B4 are rare. Most common clinical syndromes associated with Coxsackievirus B4 include aseptic meningitis, encephalitis, myopericarditis, neonatal infections, febrile rash illnesses, and respiratory manifestations. ^[9],[10] Coxsackievirus B4 has an endemic pattern of circulation. Approximately 60% of all Coxsackievirus B4 detections came from young infants, and CSF was the most common source. Similar to Coxsackievirus B5, the summer-fall seasonality in Coxsackievirus B4 detections was more prominent than for the majority of other serotypes. Coxsackievirus B4 had one of the highest proportions of reports with fatal outcome (9.8%), with a significantly higher risk of death when compared with fatal outcomes among persons infected with any other enterovirus serotype.

Coxsackievirus B3 has been commonly associated with myopericarditis, aseptic meningitis, neonatal systemic illness, meningoencephalitis in immunodeficient persons, herpangina, and fever with rash. ^[11] Outbreaks of neonatal infections and herpangina and a cluster of myocarditis cases in the context of community wide Coxsackievirus B3 outbreak have been reported. ^[12] Coxsackievirus B3 has an epidemic pattern of circulation. The proportion of children aged <1 year among reports of Coxsackievirus B3 detections exceeded 60%, and respiratory specimens were the most common source of detection.

Coxsackievirus B1 has been associated with outbreaks of aseptic meningitis and pleurodynia. ^[13],[14] Common clinical presentations are similar to other group B Coxsackieviruses and include aseptic meningitis, meningoencephalitis, myocarditis, hand, foot, and mouth disease, and pleurodynia. Coxsackievirus B1 also can cause systemic neonatal illness sometimes presenting as fulminant hepatitis with coagulopathy, a syndrome usually associated with echovirus 11 rather than with encephalomyocarditis syndrome typical for Coxsackieviruses B2-B5. ^[13] The summer-fall seasonality in Coxsackievirus B1 detections was more prominent than for the majority of other serotypes. ^[15]

The Coxsackie B virus genome is a single-stranded RNA molecule, approximately 7500 nuleotides long, of positive polarity. An approximately 750-nucleotide 5′-untranslated region (5′-UTR) is followed by a long, open reading frame coding for an approximately 2100-aminoacid polyprotein. This is followed by a short 3′-untranslated region (3′-UTR) and a poly (A) tail. Enteroviruses use an error-prone RNA-dependent RNA polymerase enzyme for their replication; hence, the mutation rate is very high. The 5′-UTR seems to be extremely conserved among enteroviruses because the secondary structures in this region, the cloverleaf and internal ribosome entry site, are required for efficient replication and translation of the viral RNA. ^[16] Therefore, the 5′-UTR has been used extensively in diagnostic RT-PCR assays for enterovirus infection and several important functions related to it; so, this region has been used in this study.

The type-specific diagnosis of enterovirus infection still relies on neutralisation assays using pools of type-specific polyclonal antisera ^[4],[17] followed by confirmation with monospecific antisera, but the results are often difficult to interpret. Recent developments in molecular biology have enabled the detection of Coxsackie B virus genomes directly in clinical samples. These tests do not, however, allow typing of the virus strains, which would be very useful for both epidemiological and clinical purposes. It is, therefore, valuable to obtain a type-specific enterovirus molecular method for diagnosis, focussing on the regions that remain relatively conserved during viral replication. The high sensitivity and specificity of the RT-PCR followed by RT-LAMP assay have made it to be very useful for diagnosis of Coxsackie B viruses.

Both the above antigenic and molecular methods depend upon the prior isolation of Coxsackieviruses in cell culture, a process that typically requires 1-3 weeks. Because many clinical specimens contain heterotypic mixtures, further separation procedures are needed before intratypic identifications can be performed. The routine diagnostic methods for enteroviruses by virus cell culture are followed by serum neutralisation test, the gold standard for enterovirus typing. This method is generally reliable, but also labour-intensive, time-consuming, and costly. Furthermore, the supply of antisera is limited and the problem of "untypeable" enteroviruses is frequently encountered. The comparatively slower procedures of in vivo amplification of Coxsackieviruses in the cell culture may be replaced by the rapid in vitro amplification of viral RNA sequences by the LAMP.

In seeking such diagnostic specificity, we investigated the applicability of a novel nucleic acid amplification method, LAMP. ^[1] LAMP amplifies DNA with high specificity, efficiency, and speed under isothermal conditions. The LAMP reaction requires a DNA polymerase with strand displacement activity and a set of four specially designed inner and outer primers that recognise six distinct sequences on the target DNA. Only simple, cost-effective equipment amenable for use in hospital laboratories is required. This method also exhibits extremely high amplification efficiency, owing, in part, to its isothermal nature; no time is lost as a result of changes in temperature, and the reaction can be conducted at the optimal temperature for enzyme function. Moreover, the inhibition reaction that occurs at later stages of amplification, which typically confounds PCR, is less likely to occur. Therefore, LAMP constitutes a potentially valuable tool for the rapid diagnosis of infectious diseases, such as those caused by enterovirus, in commercial or hospital laboratories. The goal was to establish a highly sensitive and species-specific LAMP-based Coxsackievirus group B amplification method and to examine its reliability in discriminating among species.

The amplification efficiency of the RT-LAMP method is extremely high due to continuous amplification under isothermal conditions, which results in the production of a large amount of target DNA as well as a large amount of the by-product magnesium pyrophosphate, which leads to turbidity. ^[18] Therefore, quantitative detection of gene amplification is possible by real-time monitoring of the turbidity in an inexpensive photometer. In addition, the higher amplification efficiency of the RT-LAMP method enables simple visual observation of amplification with the naked eye under a UV lamp in the presence of an intercalating dye, such as SYBR Green I or ethidium bromide. Thus, the RT-LAMP assay has emerged as a powerful gene amplification technique for rapid identification of microbial infections. ^[19],[20] In the present study, a one-step, single-tube, real-time, accelerated RT-LAMP assay has been standardised by targeting the 5′-UTR gene for rapid and real-time detection of group B Coxsackieviruses.

References

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