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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgements
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 32  |  Issue : 2  |  Page : 124-129
 

Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification


1 Department of Obstetrics and Gynecology; Department of Endocrinology and Metabolism, Shengjing Affiliated Hospital of China Medical University, Shenyang 110004, China
2 Department of Endocrinology and Metabolism, 1st Affiliated Hospital of China Medical University, Shenyang 110001, China
3 Department of Medical Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University; Liaoning Center for Disease Control and Prevention, Shenyang 110005, China
4 Department of Medical Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, Shenyang 110001, China

Date of Submission04-Mar-2013
Date of Acceptance27-Oct-2013
Date of Web Publication2-Apr-2014

Correspondence Address:
C An
Department of Medical Microbiology and Parasitology, College of Basic Medical Sciences, China Medical University, Shenyang 110001
China
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Source of Support: This work was supported by the National Natural Science Foundation of China (NSFC 81200653) and Natural Science Funds of Liaoning province, China (2011225020 and 20111108)., Conflict of Interest: We declare that we have no potential confl icts of interest relevant to this article.


DOI: 10.4103/0255-0857.129779

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 ~ Abstract 

Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Results : A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. Conclusions : The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.


Keywords: Clinical specimens, Enterovirus 71, hand foot and mouth disease, reverse transcription loop-mediated isothermal amplification


How to cite this article:
Wang D, Wang X, Geng Y, An C. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification. Indian J Med Microbiol 2014;32:124-9

How to cite this URL:
Wang D, Wang X, Geng Y, An C. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification. Indian J Med Microbiol [serial online] 2014 [cited 2019 Oct 15];32:124-9. Available from: http://www.ijmm.org/text.asp?2014/32/2/124/129779



 ~ Introduction Top


Hand foot and mouth disease (HFMD) is a severe infective disease among young children. Several new epidemics of HFMD have broken out around the world [1],[2] and a tendency of outbreak seems to rise significantly in China mainland in the recent years. [3],[4] The data from Ministry of Health of China have indicated that HFMD is a quite common infectious disease of class C since 2008 (Official website of Chinese Centre for Disease Control of the People's Republic of China). HFMD can be caused by many species and different types of human enterovirues, such as human enterovirus 71 (EV71), coxsackievirus A16, A4, A5 (CVA16, CVA4, CVA5), etc., However, the EV71 has been recently identified as the primary and major pathogen of HFMD among young children, and could result in severe neuphrotic syndrome, even fatal. [5],[6]

Until now, the diagnosis of HFMD is meanly dependent on the clinical symptoms, detection of virus-specific antibodies or virus culture and identification. But, the serodiagnosis is easy to cross-response with other pathogen and the process of virus identification is time-consuming. Reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR have been recently reported to detect EV71 with high sensitivities and specificity. [7],[8] However, these methods are limited in clinical applications because of the expensive equipment and complicated operation process. Therefore it is essential to develop a sensitive, specific and rapid approach to diagnose HFMD for an early treatment.


 ~ Materials and Methods Top


Samples

A total of 116 specimens (from 116 patients), including 88 stool samples and 28 throat swabs were collected from clinically suspected HFMD patients in Liaoning province of China during February to October, 2009. The patients were selected following the guidelines for HFMD diagnosis from the Chinese Centre for Disease Control (China CDC) and the patients suffered from some typical symptoms of the disease, such as febricity, maculopapule and vesicular on the skin of hands and feet or palms and soles. All the patients are aged 1-6 years; the throat swabs and stool samples were collected on the day of admission. All samples were collected between 2 and 5 days after the onset of illness. Samples were stored in 3-5 ml of preservation solution (Hanks solution containing 10 μg/ml gentamicin and 0.25 μg/ml amphotericin B) at −70°C until the genomic viral RNA was extracted.

The study was conducted in accordance with the amended declaration of Helsinki and was approved by the Provincial CDC Review Board. The clinical samples used in this study were appropriately coded to be anonymous, and guidelines were followed for use of clinical material and access to diagnostic results.

RNA extraction

Total RNA was isolated from 100 μl viral culture supernatant or 0.2 g of stool sample, which was suspended in 1.0 ml phosphate buffered saline (PBS) buffer, or 100 μl aliquot swab specimens from each patient, using RNeasy mini kit (QIAGEN[TAG:2][/TAG:2]

Co., Ltd, Shanghai, China) following the instructions of manufacture. Total RNA were eluted in a final 60 μl of RNase-free water and stored at −80°C.

RT-PCR

The primers of RT-PCP for EV71 were 5′- GCAGCCCAAAAGAACTTCAC-3′ and 5′- ATTTCAGCAGCTTGG

AGTGC-3′. Conventional RT-PCR was carried out as described by the one-step RT-PCR detection kit (Promega Beijing Biotech Co. Ltd, Beijing, China). All the protocols refer to "Guide for Control and Prevention of HFMD" issued by the Ministry of Public Health of China (China Ministry of Health, 2010). The 50 μl of mixture containing extracted RNA 5 μl, 10 × buffer 5 μl, dNTP 2 μl, 5 × enhancer 10 μl, Quant RTase 0.5 μl, Hotmaster Taq 2.5 μl, Rnasin 0.5 μl and EV71-S and EV71-A primer each 1 μl (20 pmol). The reaction was performed for 30 min at 50°C, 2 s at 45°C, 30 s at 70°C with 32 cycles of amplification, extensions at 72°C for 10 min. The product of amplification was checked by 1.5% agarose gel electrophoresis and EB staining.

Real-time PCR

The real-time PCR were carried out in the Light Cycler 480 (Roche, real time thermal cycler), according to the instruction of EV 71 fluorescence PCR detection kit (Da An Gene Co., Ltd. of Sun Yat-sen University, Guang Zhou, China). Briefly, total 20 μl mixture containing template RNA 5 μl, Taq DNA polymerases 1 μl, reverse transcriptase 1 μl, forward primer 0.2 μM and 0.2 μM reverse primer. The reaction was performed for 20 min at 42°C, 2 min at 93°C, following 40 cycles amplification of 95°C 30s, 55°C 45 s and 72°C, 30 s for denaturation, annealing and extension steps, respectively.

Design of reverse-transcription loop-mediated isothermal amplification primers

DNA Star was employed to analyse the gene nucleotide sequence of EV 71 VP1 from GenBank (GQ121432). Three sets of universal primers, including inner primers (FIP, BIP), outer primers (F3, B3) and loop primers (Loop F, Loop B) targeting highly conserved immunogenic regions of VP1 were selected with the primer design software (LAMP Primer Explorer 4, Eiken Chemical Co. Ltd). The specificity of the primers was analysed online BLAST [Table 1]. The primers were synthesised by Sangon Biotech Co. Ltd., Shanghai, China.
Table 1: The sequences and genomic position of primers

Click here to view


RT-LAMP assay

Total 25 μl amplification system consists of RNA template 2.5 μl, Bst DNA polymerase (8 U/μL, NEB Inc.) 1 μl, Bst DNA polymerase buffer (10×) 2.5 μl, AMV inverse transcriptase (10 U/μl, Promega Beijing Biotech Co.Ltd. Beijing, China) 1 μl, dNTP (10 mmol/μl) 1 μl, Betaine (5 mol/L, Sigma) 5 μl, MgSO 4 ( 100 nmol/μl) 1.5 μl, RNase Inhibitor 0.5 μl, six primers corresponding to VP1 gene 1 μl each (5 pmol each of outer primers F3 and B3, 40 pmol each of inner primers FIP and BIP, 20 pmol each of loop primers LB and LF, respectively) and H 2 O 4 μl. The reaction mixture was incubated at 64°C for 60 min in thermal cycler. The products were analysed by agarose gel and capillary electrophoresis.

Specificity of RT-LAMP assay

The specificity of the assay was evaluated by cross-reactivity tests with RNA extracted from EV 71 PV1, Coxsackie virus A16, Rotavirus, Norovirus, Sapovirus and Astrovirus. All of the virus positive samples were confirmed by RT-PCR and kindly given as a present by Center for Disease Prevention and Control of Liaoning province of China.

Sensitivity of RT-LAMP assay

The EV 71 PV1 was cultured in vero cells and the concentration of virus was determined by plaque assay. The sensitivity and the lowest detection limit of RT-LAMP assay was tested with various concentrations of virus RNA, which ranged from 10 5 to 0.1 PFU/ml in a serial 10-fold dilution (1.0 × 10 -1 -1.0 × 10 -6 ). The sensitivity of[TAG:2][/TAG:2]

RT-LAMP assay was simultaneously compared with real-time PCR and RT-PCR.

Statistical analysis

Statistically significant of this study was evaluated by using Chi-square test. The agreement analysis between different assays was measured. A significant difference was considered at the P value of less than 0.05.


 ~ Results Top


Specificity of RT-LAMP for EV 71 detection

The products of amplified were analysed by1.5% agarose gel electrophoresis and capillary electrophoresis. The specific nucleic acid of EV 71 can be observed as a series of disparate long strand DNA. The ladder-like mixture of stem-loop DNAs was consistent with expected. The RT-LAMP of EV 71 -specific assay did not show any cross reactivity with Coxsackievirus A16, Rotavirus, Norovirus, Sapovirus or stellavirus [Figure 1]. Similar results also can be proved by capillary electrophoresis [Figure 2].
Figure 1: Specifi city of RT-LAMP for Human Enterovirus 71 The specifi c product of EV71 nucleic acid specimen can be observed as a series of disparate length macromolecular, long strand DNA in the 1.5% agarose gel electrophoresis. The ladder-like mixture of stem-loop DNAs was consistent with theoretical results. The RT-LAMP primers for EV71 did not show any specifi c bands or cross reactivity of Coxsackievirus A16, Rotavirus, Norovirus, Sapovirus or stellavirusis.

Click here to view
Figure 2: Specifi city detection of Human Enterovirus 71 gene by RT-LAMP with Capillary electrophoresis. Capillary electrophoresis was used to analyse the amplifi cation products performed by RT-LAMP. Nucleic acid of EV71 showed (1) a series of disparate length macromolecular. The ladder-like mixture of stem-loop DNAs was consistent with theoretical consequence. RT-LAMP primers for EV71 did not show any specifi c bands or cross reactivity of Coxsackievirus A16 (2), Rotavirus (3), Norovirus (4), Sapovirus (5) and negative
control (6). M: 50 bp Marker.


Click here to view


Sensitivity of RT-LAMP for EV 71 detection

The typical amplified bands were observed for 10 -1 -10 -5 serial dilution of specimen with signals gradually weaker. The minimum limit detection by RT-LAMP assay was 0.01PFU ([Figure 3]A, Real time monitoring of RT-LAMP see Supplemental [Figure 1] and [Figure 2]). The minimum limit of RT-PCR and real-time PCR was about 0.1PFU [Figure 3]B and C].
Figure 3: Sensitivity of three methods for detection EV71 (A) Sensitivity of RT-LAMP for detection EV71, the detection limit is 0.01PFU, (B) Sensitivity of RT-PCR for detection EV71, the detection limit is 0.1PFU, (C) Sensitivity of real-time PCR for detection EV71, the detection limit is 0.1PFU

Click here to view






RT-LAMP assay from clinical samples

RT-LAMP, RT-PCR and real-time PCR were performed to analyse EV 71 for 88 stool samples and 28 throat swabs [Figure 4]. Comparative analysis of positive detection rates was carried out with RT-LAMP and compared with RT-PCR or real-time PCR, respectively. The results showed that the positive detection rate of RT-LAMP for EV 71 gene was significantly higher than RT-PCR and real-time PCR (χ2 = 6.20, P X2 0.05). The agreement analysis between different assays showed that Kappa = 0.188, P = 0.036 between RT-LAMP and real-time PCR assays, Kappa = 0.438, P = 0.000 between RT-LAMP and RT-PCR assays.
Figure 4: Comparison of detection rates with three assay methods from clinical samples Total of 116 clinical samples were collected from suspected HFMD infant patients. The assay results exhibited a higher detection rate by RT-LAMP (56.9%, 66/116) than real-time PCR (43.1%, 50/116) and RT-PCR 34.5%, 40/116), (P < 0.05).

Click here to view


The concordance and discordance of three assay methods for 36 cell culture positive samples, 28 from stool samples and 8 from swab samples, as shown in [Table 2].
Table 2: The three detected methods for cell culture positive samples

Click here to view



 ~ Discussion Top


HFMD caused by EV71 is an emerging worldwide epidemic infection most commonly occurring in young children, especially pre-school-aged children. [1],[2],[3],[4],[5],[6] EV71 can cause severe neurological disease with complex transmission routes, rapid progression and high mortality. There is no vaccine or specific anti-viral drugs against EV71 infection. Laboratory detection methods include viral isolation from cell cultures, serum antibody assay, nucleic acid detection by RT-PCR. The time-consuming cell culture analysis is complicated. Serological measurement cannot refrain from the effect of enterovirus cross-reaction, and thus neither method is suitable for emergency diagnosis at the earlier stage of HFMD. Even though RT-PCR is one of the most rapid and available systems known today, 6-7 hours is needed for results to be available, so it is difficult to meet the needs of emergency diagnosis. In case of real-time PCR, despite the advantage of sensitivity, specificity and quickness, the cost is so high that it may not be readily applicable in the detection of the virus in clinical specimens. Therefore, early detection and rapid diagnosis is critically important on prevention, treatment and outcome of the disease, especially in developing countries.

In this study, we developed a RT-LAMP, in which, the three pairs of primers can identify and complement eight distinct regions of the target sequences. Moreover, with the joint action of inverse transcriptase and Bst DNA polymerase, the target template can then be isothermal- amplified in 1 hour. The detection of the EV 71 from the clinical samples with the RT-LAMP assay revealed that the method is highly specific, sensitive and efficient, and minimises cross-contamination due to its one-step operation. The RT-LAMP assay thus seems to be a promising method that might be applied widely in clinical routine detection.

The key point of the LAMP technique is its primers design. The gene region coding VP1 protein was selected and focused in this study. VP1 is a major coat protein with antigenic sites contributing to type specificity. Several studies have indicated the potential of VP1 protein to act as an antigen in the diagnosis for EV71. [9],[10] The three sets of primers in this domain can specifically identify eight definite regions of the target sequence. Furthermore, when the six primers match strictly with target sequence, amplification becomes available, and thus the reaction is not easily influenced by the non-target sequence. This phenomenon guarantees the specificity of the technique. The results in this study demonstrated the specificity of RT-LAMP for EV 71 detection.

In case of stool samples and throat swabs with relatively low viral loads, analysis results demonstrated RT-LAMP assay has 10 times sensitivity and higher positive detection rate than real-time PCR and RT-PCR. The detection procedure is simple, rapid and does not require any special equipment and trained operators. RT-LAMP is convenient and thus its application will be appropriate and popular as a routine diagnostic method for EV71 infection in primary medical centre or hospitals. Optimisation and improvement of the RT-LAMP system may implement multiple detections, that is, carrying out one reaction could lead to the detection of multiple HFMD relative viruses. This would be useful and promising in early diagnosis, prevention and control of HFMD.

HFMD is mostly caused by human EV71 and coxsackievirus A16 (CA16), although several other human enteroviruses including coxsackieviruses CA2, CA4-CA7, CA10, CA12, and CB1-CB5 can also cause the disease. More than 60% of the HFMD caused by EV71, approximately 20% by CA16 and other enteroviruses accounted for 10%, respectively, in China. [5] Similar results in present study, the detective rates are 63.89%, 72.22% and 77.78% by the RT-PCR, real time PCR and RT-LAMP, respectively, in cell culture positive samples, the negative cases may be infected by the CA16 or other human enterovirus.

We also noticed there were several publications on the evaluation of the LAMP assay for the detection of EV71 in the region of China, but all of the cases were from south of China, our cases were from north-east of China. And we used different sequences in VP1 to attempt getting more sensitive and specific result compared with other researches. Moreover, in our research we compared the samples of stool and throat swab by the three assays and proved RT-LAMP is effective in detecting both stool and throat swab samples.

In conclusion, the detecting rate of RT-LAMP is higher, the protocol is easy to operate and time saving, which can be finished in single condition within 60 min, and no expensive instrument was needed. It can be applied for rapid diagnosis of the disease, especially in poor areas or developing countries.


 ~ Acknowledgements Top


The authors thank all of the members of our laboratories for their helpful assistance.

 
 ~ References Top

1.Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 2010;10:778-90.  Back to cited text no. 1
    
2.Ryu WS, Kang B, Hong J, Hwang S, Kim J, Cheon DS. Clinical and etiological characteristics of enterovirus 71-related diseases during a recent 2-year period in Korea. J Clin Microbiol 2010;48:2490-4.  Back to cited text no. 2
    
3.Yang F, Ren L, Xiong Z, Li J, Xiao Y, Zhao R, et al. Enterovirus 71 outbreak in the People's Republic of China in 2008. J Clin Microbiol 2009;47:2351-2.  Back to cited text no. 3
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4.Hu, XY, Zheng X, Wen ZF. Investigation of epidemiology of hand-foot-mouth disease in 2346 patients. Chin J Nosocomiol 2012;22:2333-5.  Back to cited text no. 4
    
5.Ni H, Yi B, Yin J, Fang T, He T, Du Y, et al. Epidemiological and etiological characteristics of hand, foot, and mouth disease in Ningbo, China, 2008-2011. J Clin Virol 2012;54:342-8.  Back to cited text no. 5
    
6.Deng AP, Zhang YH, Sun LM. Survey on the recessive infection of pathogen to hand-foot-mouth disease among healthy adults and children in Guangdong province. Zhozhonghua Liu Xing Bing Xue Za Zhi 2012;33:189-91.  Back to cited text no. 6
    
7.Chen TC, Chen GW, Hsiung CA, Yang JY, Shih SR, Lai YK, et al. Combining multiplex reverse transcription-PCR and a diagnostic microarray to detect and differentiate enterovirus 71 and coxsackievirus A16. J Clin Microbiol 2006;44:2212-9.  Back to cited text no. 7
    
8.Chiang PS, Huang ML, Luo ST, Lin TY, Tsao KC, Lee MS.Comparing molecular methods for early detection and serotyping of enteroviruses in throat swabs of pediatric patients. PLoS One 2012;7:e48269.  Back to cited text no. 8
    
9.Oberste MS, Maher K, Kilpatrick DR, Pallansch MA. Molecular evolution of the human enteroviruses: Correlation of serotype with VP1 sequence and application to picornavirus classification. J Virol 1999;73:1941-8.  Back to cited text no. 9
    
10.Shih SR, Li YS, Chiou CC, Suen PC, Lin TY, Chang LY, et al. Expression of capsid proteinVP1for use as antigen for the diagnosis of enterovirus71 infection. J Med Virol 2000;61:228-34.  Back to cited text no. 10
    


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