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1、ORIGINAL ARTICLE Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): a general methodology for detecting rapidly mutating viruses Andreas V. HadjinicolaouGabriella A. FarcasVictoria
2、L. Demetriou Tony MazzulliSusan M. PoutanenBarbara M. WilleyDonald E. Low Jagdish ButanySylvia L. AsaKevin C. KainLeondios G. Kostrikis Received: 10 September 2010/Accepted: 6 December 2010/Published online: 11 January 2011 ? Springer-Verlag 2011 AbstractEmerging infectious diseases have caused a gl
3、obal effort for development of fast and accurate detection techniques. The rapidly mutating nature of viruses presents a major diffi culty, highlighting the need for specifi c detection of genetically diverse strains. One such infectious agent is SARS-associated coronavirus (SARS-CoV), which emerged
4、 in 2003. This study aimed to develop a real-time RT-PCR detection assay specifi c for SARS-CoV, taking into account its intrinsic polymorphic nature due to genetic drift and recombination and the possibility of continuous and multiple introductions of genetically non-identical strains into the huma
5、n population, by using mismatch-tol- erant molecular beacons designed to specifi cally detect the SARS-CoV S, E, M and N genes. These were applied in simple, reproducible duplex and multiplex real-time PCR assays on 25 post-mortem samples and constructed RNA controls, and they demonstrated high targ
6、et detection ability and specifi city. This assay can readily be adapted for detection of other emerging and rapidly mutating pathogens. Introduction Emerging viruses with a high rate of genetic drift cause setbacks in the global scientifi c effort to develop fast and accurate diagnostic detection m
7、ethods. A novel virus, SARS-associated coronavirus (SARS-CoV), which spread throughout the world in 2003, was quickly characterised 13, 21 and linked to severe acute respiratory syndrome (SARS) 2, 11, 15. Human coronaviruses are confi ned mainly to the upper respiratory tract, causing simple ill- ne
8、sses, whereas in animals, they have been associated with severe diseases 16. There is still an ongoing possibility of SARS-CoV re-emergence, since evidence suggests that it arose from an animal coronavirus 5, 25. Poor clinical defi nition and lack of known prevention methods and treatments 10, 22 me
9、an that early diagnosis is crucial. There has been a huge effort to develop diagnostics 1, 9, 12, 17, 18, 26, 27, nevertheless, molecular detection tests suffer from speed and sensitivity limitations and are not standardised 14, 22. Accurate and prompt detection of the virus is, therefore, a priorit
10、y. Current molecular assays fail to consider the intrinsi- cally polymorphic nature of coronaviruses, as seen in the pathogenic SARS-CoV Tor2 and Urbani strains, and the possibility of continuous introductions of genetically non- identical strains into human populations. This study aimed to develop
11、a real-time RT-PCR assay using mismatch-tol- erant molecular beacons 6, 7, 24 to detect SARS-CoV A. V. Hadjinicolaou ? V. L. Demetriou ? L. G. Kostrikis ( porcine epidemic diarrhea virus (PEDV), AF353511; transmissible gastroenteritis virus (TGEV), AJ271965; canine coronavirus (CCoV), D13096; feline
12、 coronavirus (FCoV), AY204704; porcine respiratory coronavirus (PRCoV), Z24675. Those from group 2 (G2) were bovine coronavirus (BCoV), AF220295; murine hepatitis virus (MHV), AF201929; human coronavirus OC43 (HCoV-OC43), M76373; porcine hemagglutinating encephalomyelitis virus (HEV), AY078417; rat
13、coronavi- rus (RtCoV), AF207551; and the references virus for group 3 (G3) was infectious bronchitis virus (IBV), M95169. Sequence alignments were performed with ClustalW 23 to identify genomic regions with the highest genetic dis- tance between SARS-CoV and other coronavirus strains, with special e
14、mphasis on probe-target sequences that encompass gaps or deletions in the SARS-CoV genome. Like the probe sequences, primer selection was also based on the ability for amplifi cation of the SARS-CoV targets that would form primer-target and probe-target hybrids with a theoretical melting temperature
15、 that would maxi- mize the ability of the primer to bind to the target even if nucleotide substitutions are present to achieve mismatch tolerance. Finally, primer sequences were selected that enable the amplicons formed to be approximately the same length, about 100 nucleotides. Internal positive co
16、ntrol A synthetic oligonucleotide was designed to be amplifi ed by theforwardSgeneandreverseEgene primers (Table 1)and usedasaninternalpositivecontrol(IPC)during amplifi cation. Thermal profi les of molecular beacons Thethermodynamiccharacteristicsofthemolecular beacons were assessed by melting curve analyses per- formed in a 7700 Prism Spectrofl uorometric Thermal Cycler (Applied Biosystems, Foster City, CA, USA) and validated using the 7900 HT Fast Real-Time PCR Sys- tem (Applied Biosystems).
