Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a highly sensitive detection of enterovirus in the stool samples of acute flaccid paralysis cases

Abstract

Background: In the global eradication program for poliomyelitis, the laboratory diagnosis plays a critical role by isolating poliovirus (PV) from the stool samples of acute flaccid paralysis (AFP) cases. In this study, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) system for a rapid and highly sensitive detection of enterovirus including PV to identify stool samples positive for enterovirus including PV.

Methods: A primer set was designed for RT-LAMP to detect enterovirus preferably those with PV-like 5'NTRs of the viral genome. The sensitivity of RT-LAMP system was evaluated with prototype strains of enterovirus. Detection of enterovirus from stool extracts was examined by using RT-LAMP system.

Results: We detected at least 400 copies of the viral genomes of PV(Sabin) strains within 90 min by RT-LAMP with the primer set. This RT-LAMP system showed a preference for Human enterovirus species C (HEV-C) strains including PV, but exhibited less sensitivity to the prototype strains of HEV-A and HEV-B (detection limits of 7,400 to 28,000 copies). Stool extracts, from which PV, HEV-C, or HEV-A was isolated in the cell culture system, were mostly positive by RT-LAMP method (positive rates of 15/16 (= 94%), 13/14 (= 93%), and 4/4 (= 100%), respectively). The positive rate of this RT-LAMP system for stool extracts from which HEV-B was isolated was lower than that of HEV-C (positive rate of 11/21 (= 52%)). In the stool samples, which were negative for enterovirus isolation by the cell culture system, we found that two samples were positive for RT-LAMP (positive rates of 2/38 (= 5.3%)). In these samples, enterovirus 96 was identified by sequence analysis utilizing a seminested PCR system.

Conclusions: RT-LAMP system developed in this study showed a high sensitivity comparable to that of the cell culture system for the detection of PV, HEV-A, and HEV-C, but less sensitivity to HEV-B. This RT-LAMP system would be useful for the direct detection of enterovirus from the stool extracts.

Figure 1

Regions in the 5'NTR of enterovirus genome examined for the design of RT-LAMP primers. a Schematic view of a model of the secondary structure of 5'NTR of enterovirus genome [22-24]. The region examined for the design of RT-LAMP primers is shown in a box. b Primary and secondary structure of 5'NTR of PV1(Mahoney) genome and RT-LAMP primers used in this study. The structure is based on the model proposed by Pilipenko et al. [22]. The region examined for RT-LAMP primers is shown in boxes on the secondary structure. The numbers on the RT-LAMP primers represent corresponding nucleotide positions on the 5'NTR. For primers that have complimentary sequence to the 5'NTR, the numbers are shown in parenthesis. c Scheme of RT-LAMP procedure examined in this study.

Figure 2

Comparison of the nucleotide sequences of enterovirus genomes examined for RT-LAMP primers. Enterovirus genomes are classified into PV-like and CBV-like 5'NTR [10,11]. The nucleotides characteristic to PV-like 5'NTR are highlighted in boxes colored by gray. Primers that have complete match for PV-like 5'NTR near and at the 3' end are presented as preferable and specific primers to PV-like 5'NTR, respectively.

Figure 3

Sensitivity and specificity of RT-LAMP system. a Sensitivity of RT-LAMP system for purified viral RNA of PV(Sabin) strains. b Kinetics of the detection in RT-LAMP system. The average time required for the detection of the signals is shown for each numbers of the copies. c Sensitivity and specificity of RT-LAMP system for enterovirus. Cell culture supernatants of the cells infected with enteroviruses were used for the detection of the viral RNA by RT-LAMP system. The numbers in the parenthesis show the titre of virus (CCID₅₀) included in the RT-LAMP reactions. The numbers of copies of the viral genome per CCID₅₀ are also shown for each virus. NT, not tested. d Sensitivity and specificity of RT-LAMP system for the viral RNA purified from stool extracts of AFP cases.

Figure 4

Comparison of the nucleotide sequences of the 5'NTR in the viral genomes of enterovirus isolates. The nucleotides characteristic to PV-like 5'NTR are highlighted in boxes colored by gray.

Figure 5

Comparison of the nucleotide sequences of the regions in the viral genomes of cVDPV and iVDPV strains examined for RT-LAMP primers. The nucleotides characteristic to PV-like 5'NTR are highlighted in boxes colored by gray.