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Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT PCR.

Perera D, Podin Y, Akin W, Tan CS, Cardosa MJ - BMC Infect. Dis. (2004)

Bottom Line: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control.When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Health & Community Medicine, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, 94300 Malaysia. davidperera@yahoo.com

ABSTRACT

Background: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.

Methods: Local strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens.

Results: A total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.

Conclusions: Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

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Related in: MedlinePlus

Alignment of HEV71 and CVA16 genomes in the binding location of primer pair 159S/162A. Dots "." indicate nucleotide identity with the primer sequence; In degenerate positions, K is G or T; R is A or G; Y is C or T. The boxed region is the position with the potential to bind to HEV71 template leading to extension and amplification.
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Figure 2: Alignment of HEV71 and CVA16 genomes in the binding location of primer pair 159S/162A. Dots "." indicate nucleotide identity with the primer sequence; In degenerate positions, K is G or T; R is A or G; Y is C or T. The boxed region is the position with the potential to bind to HEV71 template leading to extension and amplification.

Mentions: In order to understand the reason for this mispriming, we sequenced 8 local isolates of CVA16 through the primer binding sites to determine if there had been any mutations that could account for this. Very few sequences of CVA16 strains have been deposited in GenBank, and the prototype strain G-10 was used in the design of the original published set of HEV71 specific primers, 159S/162A [8]. The strains we have isolated since 1998 have not shown very much change in the primer binding regions, but a few nucleotide differences may have been critical in allowing mispriming to occur. Figure 2 shows an alignment of different genogroups of HEV71 and CVA16 prototype G-10 with the consensus sequence of local CVA16 strains in the primer binding regions. All our local CVA16 strains show complete identity with the sense primer 159S but we infer that this primer was not meant to specifically bind to HEV71 since even the prototype CVA16 G-10 had only one nucleotide difference.


Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT PCR.

Perera D, Podin Y, Akin W, Tan CS, Cardosa MJ - BMC Infect. Dis. (2004)

Alignment of HEV71 and CVA16 genomes in the binding location of primer pair 159S/162A. Dots "." indicate nucleotide identity with the primer sequence; In degenerate positions, K is G or T; R is A or G; Y is C or T. The boxed region is the position with the potential to bind to HEV71 template leading to extension and amplification.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC415548&req=5

Figure 2: Alignment of HEV71 and CVA16 genomes in the binding location of primer pair 159S/162A. Dots "." indicate nucleotide identity with the primer sequence; In degenerate positions, K is G or T; R is A or G; Y is C or T. The boxed region is the position with the potential to bind to HEV71 template leading to extension and amplification.
Mentions: In order to understand the reason for this mispriming, we sequenced 8 local isolates of CVA16 through the primer binding sites to determine if there had been any mutations that could account for this. Very few sequences of CVA16 strains have been deposited in GenBank, and the prototype strain G-10 was used in the design of the original published set of HEV71 specific primers, 159S/162A [8]. The strains we have isolated since 1998 have not shown very much change in the primer binding regions, but a few nucleotide differences may have been critical in allowing mispriming to occur. Figure 2 shows an alignment of different genogroups of HEV71 and CVA16 prototype G-10 with the consensus sequence of local CVA16 strains in the primer binding regions. All our local CVA16 strains show complete identity with the sense primer 159S but we infer that this primer was not meant to specifically bind to HEV71 since even the prototype CVA16 G-10 had only one nucleotide difference.

Bottom Line: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control.When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Health & Community Medicine, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, 94300 Malaysia. davidperera@yahoo.com

ABSTRACT

Background: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification.

Methods: Local strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens.

Results: A total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage.

Conclusions: Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

Show MeSH
Related in: MedlinePlus