Mixed cell diagnostic systems for detection of respiratory, herpes and enteric viruses
View Patent ↗The present invention generally relates to the field of diagnostic microbiology, and, more particularly, to compositions and methods for detecting and differentiating one or more viruses or other intracellular parasites present in a specimen. The present invention also provides compositions and methods to evaluate the susceptibility of organisms to antimicrobial agents.
1. A multiplex method for identifying coronavirus in a sample, comprising the steps of:
a) providing
i) a sample comprising RNA from at least one coronavirus strain, wherein each of said at least one coronavirus strain RNA comprises genomic RNA and subgenomic RNA, wherein said genomic RNA comprises a polymerase gene 1b coding region and a sequence encoding a Spike (S) glycoprotein,
ii) a plurality of primers capable of amplifying said genomic RNA and said subgenomic RNA, wherein said primers are selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11,
b) contacting said genomic and subgenomic RNA from at least one coronavirus strain with said plurality of primers, wherein a reverse transcriptase-polymerase chain reaction creates a first amplicon and a second amplicon;
c) detecting said second amplicon as comprising a coronavirus leader sequence.
2. The method of claim 1 , wherein said coronavirus is the SARS coronavirus.
3. The method of claim 2 , wherein said method further comprises detecting said first amplicon as comprising a region between said polymerase gene 1b coding region and said sequence encoding the said Spike (S) glycoprotein.
4. The method of claim 1 , wherein said sample comprises coronavirus infected cells.
5. The method of claim 1 , further comprising: c) detecting said first and second amplicon on a gel.
6. The method of claim 1 , wherein said leader sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.