Comparative Studies On Different Methods Of Nucleic Acid Extraction Of Clinical Samples Associated With Hand-foot-mouth Disease

Background:Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus, of which the major pathogens are human enterovirus 71 (HEV71) and coxsackievirus A16 (CAV16). The epidemic of HFMD has been reported in most countries and regions in the world. The obvious seasonal characteristics of HFMD could be seen, mostly in summer and fall. The main patients of HFMD were preschool children, especially children aged under 5 years. Because of the epidemic infectiousness and rapid transmission of HFMD, the rapid detection of enteroviruses was important for the control of the disease. The rapid molecular detection of HFMD relied on the extraction of the nucleic acids of pathogens. Although the traditional precipitation method and the commonly used centrifugal column method could be used for extraction of the virus nucleic acids, they were time and labor-consuming, moreover, the organic solvent might be harmful to the workers. On the contrary, the automatic magnetic extraction method had the advantages of time and labor-saving, preventing injuries, as well as the processing of large quantities of samples simultaneously.Objective:To establish the automatic magnetic extraction method for enterovirus, which has the comparable sensitivity and specificity compared with centrifugal column method.Method:Clinical samples were collected from patients with HFMD. A total of two different magnetic beads-based automatic extraction method and centrifugal column method were used for extraction of nucleic acids, followed by the detection of HEV71 and CAV16 using reverse transcription real-time fluorescence PCR method (RT-qPCR). The results of centrifugal column method were used as the reference results, and the sensitivity, specificity and consistency of the two magnetic methods (A and B)were analyzed by using SPSS 18.0.The method A means the Magnetic Viral DNA/RNA Kit from Chuangzhi Lihe (Beijing) Science and Technology Company and method B means the Magnetic Viral DNA/RNA Kit from GenMag (Changzhou) biotechnology company throughout the manuscript.Results:A total of 230 pharyngeal swabs or stool samples of young patients with HFMD were collected during the period of March to October,2014 in Hebei province. All the patients aged between 2 months to 5 years old. A total of 202 samples were detected positive by centrifugal column method, of which 124 cases were HEV-71 positive,78 were CAV-16. A total of 118 and 74 samples were detected as HEV-71, CAV-16 positive by magnetic extraction method A, respectively. A total of 121 and 75 samples were detected as HEV-71, CAV-16 positive by magnetic extraction method B, respectively. Compared with the centrifugal column method, the sensitivity, specificity and agreement of magnetic extraction method A for enteroviruses were 95%,86%,94%, respectively. The sensitivity, specificity and agreement of magnetic extraction method A for HEV-71 and CAV-16 were 95%,87% and 94%; and 96%, 80%, and 95%, respectively. The sensitivity, specificity and agreement of magnetic extraction method A for throat swabs and stool samples were 95%,89% and 95%; and 95%,78%, and 93%, respectively. Compared with the centrifugal column method, the sensitivity, specificity and agreement of magnetic extraction method B for enteroviruses were 97%,89%,96%, respectively. The sensitivity, specificity and agreement of magnetic extraction method A for HEV-71 and CAV-16 were 98%, 91% and 97%; and 96%,80%,95%, respectively. The sensitivity, specificity and agreement of magnetic extraction method B for throat swabs and stool samples were 97%,89% and 96%; and 97%,89%, and 96%, respectively. There was no statistical significance between the two different magnetic methods.Conclusion:The two different magnetic methods established in this study showed good sensitivity, specificity, and consistency compared with centrifugal column method. The two magnetic beads methods showed relatively low specificity of CAV-16, which was probably due to the settlement of automation workstation hardware equipment or relatively small size of CAV-16-positive samples. These shortcomings could be improved through the optimization of hardware system and automation program. Automatic extraction of nucleic acids can not only save time and labor, improve work efficiency, and reduce the effect of chemical reagents on the staff, but also save a lot of money while processing large quantities of samples. The method shows great potential to be used for the large-scale epidemiological investigation and emergency response to the epidemic disease.