Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Abstract

Coxsackievirus B3 (CVB3) is a member of the genus Enterovirus within the family Picornaviridae and is an important pathogen of viral myocarditis, which accounts for more than 50% viral myocarditis cases. VP1 is major capsid protein that this region has a low homology in both amino acid and nucleotide sequences among Enteroviruses. Therefore we have chosen this region for designed a set of RT-LAMP primers for CVB3 detection. For this the total RNA was extracted from 24-h post infected-HeLa cells with complete cytopathic effect (CPE), and applied to a one-step reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) using CVB3-specific primers. The optimization of RT-LAMP reaction was carried out with three variables factors including MgSO concentration, temperature and time of incubation. Amplification was analyzed by using 2% agarose gel electrophoresis and ethidium bromide and SYBR Green staining. Our results were shown the ladder-like pattern of the VP1 gene amplification. The LAMP reaction mix was optimized and the best result observed at 4mM MgSO and 60°C for 90min incubation. RT-LAMP had high sensitivity and specificity for detection of CVB3 infection. This method can be used as a rapid and easy diagnostic test for detection of CVB3 in clinical laboratories.

Rapid Molecular Diagnosis of Parechovirus Infection Using the Reverse Transcription Loop-Mediated Isothermal Amplification Technique

“…The LAMP reaction, an alternative nucleic acid ampli cation method developed by Notomi et al [4], is based on strand displacement by DNA polymerase under isothermal conditions in which the temperature range is 60 °C-65 °C [4][5][6][7]. The LAMP assay is very speci c, compared to other molecular detection methods, because four primers are necessary that recognize six speci c regions of the target gene for ampli cation [4][5][6][7]. The method generates a large amount of ampli cation products in positive samples (10 9 -to 10 10 -fold in 15-60 minutes), thereby allowing some assessment of these products with the naked eye and uorescent dye [4][5][6][7].…”

“…The LAMP assay is very speci c, compared to other molecular detection methods, because four primers are necessary that recognize six speci c regions of the target gene for ampli cation [4][5][6][7]. The method generates a large amount of ampli cation products in positive samples (10 9 -to 10 10 -fold in 15-60 minutes), thereby allowing some assessment of these products with the naked eye and uorescent dye [4][5][6][7]. Another advantage of the LAMP method is that ampli cation occurs at one temperature; therefore, it does not require a thermal cycler [4][5][6][7].…”

“…The method generates a large amount of ampli cation products in positive samples (10 9 -to 10 10 -fold in 15-60 minutes), thereby allowing some assessment of these products with the naked eye and uorescent dye [4][5][6][7]. Another advantage of the LAMP method is that ampli cation occurs at one temperature; therefore, it does not require a thermal cycler [4][5][6][7]. The RT-LAMP assay, which is carried out with a RT step and a LAMP reaction in a single tube, is a simple, highly speci c, rapid, and cost-effective method [6,7].…”

“…Another advantage of the LAMP method is that ampli cation occurs at one temperature; therefore, it does not require a thermal cycler [4][5][6][7]. The RT-LAMP assay, which is carried out with a RT step and a LAMP reaction in a single tube, is a simple, highly speci c, rapid, and cost-effective method [6,7]. Fewer operating steps in RT-LAMP is also advantageous in preventing contamination, and thus being safe for technicians.…”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…Addition of SYBR green I to reaction tubes containing LAMP products lead to visible colour change from reddish orange to yellowish green, and fluorescent under UV light. SYBR green I has been used for detection of coxsackievirus (Monazah et al 2017), Plasmodium vivax and Plasmodium falciparum (Singh et al 2017), Mycoplasma synoviae (Kursa et al 2015), fish pathogen Nocardia salmonicida (Xia et al 2015), maize chlorotic mottle virus (Chen et al 2017b) and tomato chlorosis virus . Despite reported with good sensitivity, SYBR green I dye could inhibit LAMP reaction if the dye is added before isothermal incubation.…”