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Published on: July 15, 2019
A diagnostic kit for the enteroviruses Coxsackie A6 and A10
1Department of Preventive Medicine, Fuzhou Center for Disease Control and Prevention, Taijiang, Fuzhou, China.
Abstract:
Recently, there has been an upward trend in the occurrence of hand-foot-mouth disease, which is correlated with Coxsackie A6 and A10 infections. Although two separate diagnostic reagents are available for these two viral strains, the protocol and diagnosis efficiency still need to be improved. More importantly, as co-infection with these viruses is common, the development of a single test kit that can diagnose both viruses would be most beneficial for clinical practice. In our study, specific primers targeting viral nucleic acids were designed and modified. Viral nucleic acids were extracted from fecal or throat swab samples by ultrasonic rupture and silicon membrane purification. The consistency, specificity, and sensitivity of the tests were further optimized by adjusting the polymerase chain reaction (PCR) conditions. The efficiency of viral nucleic acid extraction was significantly enhanced by the ultrasonic rupture and silicon membrane elution approach. Specific amplifications of both viral nucleic acids were achieved using modified primers. The optimal conditions for PCR were also determined (60°C for 30 min and 95°C for 2 min, followed by 40 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 60°C, and elongation for 50 s at 72°C). Amplified products were confirmed as viral specific nucleotides by agarose gel electrophoresis and sequencing. The minimal nucleic acid concentration required for detection was 0.2 ng/L, which was adequate to yield satisfactory specificity and consistency. This novel diagnostic method has many advantages, including rapid protocols and accurate results, and can be promoted for large-scale clinical trials.
Insights
A new diagnostic method enables simultaneous detection of Coxsackie A6 and A10 viruses, common causes of hand-foot-mouth disease. This approach enhances diagnostic efficiency and accuracy for co-infections in clinical settings.
Area of Science:
- Virology
- Molecular Diagnostics
- Public Health
Background:
- Hand-foot-mouth disease (HFMD) incidence is rising, frequently linked to Coxsackie A6 and A10 infections.
- Current diagnostic methods for these viruses involve separate reagents, necessitating protocol and efficiency improvements.
- Co-infections with Coxsackie A6 and A10 are common, highlighting the need for a single, efficient diagnostic test.
Purpose of the Study:
- To develop a novel, single diagnostic test kit for simultaneous detection of Coxsackie A6 and A10 viral strains.
- To optimize viral nucleic acid extraction and polymerase chain reaction (PCR) conditions for enhanced diagnostic efficiency and accuracy.
Main Methods:
- Designed and modified specific primers targeting Coxsackie A6 and A10 viral nucleic acids.
- Extracted viral nucleic acids from fecal or throat swab samples using ultrasonic rupture and silicon membrane purification.
- Optimized PCR conditions, including denaturation, annealing, and elongation temperatures and times, and confirmed amplified products via agarose gel electrophoresis and sequencing.
Main Results:
- Achieved efficient viral nucleic acid extraction using ultrasonic rupture and silicon membrane elution.
- Demonstrated specific amplification of both Coxsackie A6 and A10 viral nucleic acids with modified primers.
- Determined optimal PCR conditions and established a minimal detection concentration of 0.2 ng/L with high specificity and consistency.
Conclusions:
- Developed a novel, rapid, and accurate diagnostic method for simultaneous detection of Coxsackie A6 and A10.
- The new method offers improved efficiency and accuracy for diagnosing HFMD, particularly in cases of co-infection.
- This diagnostic approach is suitable for large-scale clinical trials and widespread clinical application.
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