Establishment Of Isothermal Nucleic Acid Amplification Assays For The Detection Of MERS-CoV And HRVs

Viral infectious diseases, especially those can cause respiratory tract infections, have long been major concerns in the field of public health globally. On the one hand, the re-emergence of some well known viruses, such as West Nile virus (WNV) and Ebola virus (EboV), caused outbreaks. On the other hand, the occurrence of newly emerging ones like Middle East respiratory syndrome coronavirus (MERS-CoV) and influenza A H7N9, contributes to the increasing global concern. In addition, the lack of effective drugs or vaccines makes the application of reliable and rapid detection methods an important measure for their control and prevention. Currently, the polymerase chain reaction (PCR) technology has been the main platform for molecular diagnosis of microbial infections. However, those PCR-based assays need fine and expensive instruments as well as long reaction time. These requirements make it unsuitable to resource-limited clinics and quarantine sites. In comparison, isothermal nucleic acid amplification techniques such as loop mediated isothermal amplification (LAMP), genome exponential amplification reaction (GEAR) and isothermal multiple-self-matching-initiated amplification (IMSA) have the ability to be performed with simple equipments and are more applicable to primary clinical settings.First of all, we established three detection methods based on the above-mentioned three isothermal amplification techniques for MERS-CoV, a novel coronavirus. The methods targeted Nucleocapsid (N) gene and were carried out under isothermal conditions at 63?. The products were detected by collecting fluorescence signals or visual inspection of color change. The specificities of the assays were validated by detecting several human Coronaviruses and common respiratory viruses and the cross-reactivity was not observed. With in vitro transcribed RNA as templates, the limit of detection (LoD) of each assay was evaluated and compared to that of a real time reverse-transcription PCR (rRT-PCR) assay recommended by Centers for Disease Control and Prevention. The results showed that the real-time RT-LAMP and RT-GEAR had equal LoD (5 X 102 copies/reaction) while the LoD of RT-IMSA was 102 copies/reaction. The LoD of visual RT-IMSA was 5×102 copies/reaction, while the LoDs of visual RT-LAMP and RT-GEAR were 103 copies/reaction. Experiments to detect MERS-CoV using spiked pharyngeal swabs inoculated with pre-quantified MERS-CoV like particles were also carried out and the results showed the sensitivities of the real-time RT-LAMP and RT-GEAR were 104 copies/reaction. And the sensitivity of the real-time RT-IMSA was 103 copies/reaction, better than those of the above two methods. Therefore, the established assays have the potential to be widely adopted as platforms for simple and rapid detection of MERS-CoV infections and are suitable to resource-limited clinical sites.In the second part, a reverse-transcription genome exponential amplification reaction (RT-GEAR) assay was developed for rapid and universal detection of human rhinoviruses. The assay employed a set of four primers that recognized sequences of 5'-untranslated region (UTR) gene for amplification at 65? for 60 min with a real-time fluorometer (Genie(?) ?; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses or respiratory viruses. With in vitro transcribed RNA containing the amplification regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5,50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples and the results were compared with those obtained by a published semi-nested PCR assay followed by sequencing. The results showed that the sensitivity and specificity of the pan-HRVs RT-GEAR assay were 98.08%and 100%, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(?)? fluorometer was demonstrated to a sensitive, specific, rapid and field-friendly assay for the universal detection of HRVs infection.inIn conclusion, three isothermal nucleic acid amplification assays were established to detect the novel coronavirus, MERS-CoV. They provide rapid and effective methods for the detection of MERS-CoV and are of great use in quarantine and clinical settings. They are important preparations to deal with possible outbreaks in China and suitable to field use and resource-limited clinical settings. Furthermore, a RT-GEAR assay was developed to detect pan-HRVs, which are the most prevalent human respiratory viruses. The RT-GEAR assay has the ability to be applied to all the three species A, B and C and is therefore a universal, reliable and rapid tool for the detection of HRVs. It contributes to the early diagnosis of HRVs-associated diseases and prevent the occurrence of more serious diseases.