Establishment Of Methods For Rapid Detection Of Important Respiratory Viruses

Respiratory viruses are a series of pathogens that can evoke respiratory system or other systems diseases by the respiratory tract. Its mode of transimission determine the chance of world-wide pandemic, such as Influenza or SARS. Rapid detection of these viruses is critical to take effective precaution and therapeutic measure.This study developed Multiplex PCR to detect important respiratory viruses. Multiplex Polymerase chain reaction is a developed PCR method that can detect many pathogens simultaneously by running many distinct gene amplifications. Respiratory viruses which consist of many species cause similar clinical symptom, so, this method is suitable to detect and diagnose the infection of this kind of viruses. A multiplex Polymerase chain reaction(PCR) method was developed and optimized to simultaneously detect Influenza A virus (IA),Respiratory syncytial virus(RSV)and SARS coronavirus(SARS-CoV). Cultured IA,RSV and SARS-CoV were detectable to sensitivities of 10^1.7TCID₅₀/ml,10TCID₅₀ /ml and 10^1.5 TCID₅₀/ml. Meanwhile, we developed another multiplex PCR to detect three important serotypes of adenovirus(Ad),namely Ad₂ Ad₃ and Ad₇. Cultured Ad₂ Ad₃ and Ad₇ were detectable to sensitivities of 0.01～1TCID₅₀/ml. The multipoex PCR assay may be completed within 6 hours, and used as a rapid method for the differentiation of these respiratory viruses.Real-time PCR have good quality of saving time , reducing carry-over contamination and quantitating nucleic acid of the viruses, It has been widely used in the field of the clinical detection when the cost of this method lowered gradually. The Multiplex real-time PCR make less cost because of saving the enzyme and fluorescent dye or probe . This study used SYBR GREEN I to detect IA,RSV and SARS-CoV. Cultured IA,RSV and SARS-CoV were detectable to sensitivities of 10^0.7TCID₅₀/ml,1TCID₅₀ /ml and 10^-0.5 TCID₅₀/ml , The result showed that this approach had higher sensitivity than the multiplex PCR and satisfactory specificity like that. SARS-CoV need detecting in BSL-3 laboratory, so we applied the method of IFA to detect the antibody of SARS-CoV based on developing cloned cell strain expressing specific antigen. Constructing BHK-21 cell strain expressing N protein of SARS-CoV can avoid laboratory infection and restriction of deficiency of BSL-3 laboratory by IFA . Meanwhile, this method will be completed in 3 hours, so it provide a rapid and secure means to detect the infection of SARS-CoV.