Selective detection of human rhinovirus
View Patent ↗A process for detecting human rhinovirus nucleic acid in a biological sample, includes producing an amplification product by amplifying an human bocavirus nucleotide sequence using a forward primer of SEQ ID NO: 1, and a reverse primer of SEQ ID NO: 2, and measuring said amplification product to detect human rhinovirus in said biological sample. Also provided are reagents and methods for detecting and distinguishing human rhinovirus from other viruses. A kit is provided for detecting and quantifying human rhinovirus in a biological sample.
1. A process of detecting human rhinovirus in a biological sample comprising: producing an amplification product by amplifying a human rhinovirus nucleotide sequence using a forward primer homologous to a region within 356-563 of human rhinovirus, and a reverse primer homologous to a region within 356-563 of human rhinovirus, under conditions suitable for a polymerase chain reaction; and measuring said amplification product to detect human rhinovirus in said biological sample, wherein said forward primer is the isolated nucleotide having the sequence 5′-CPXGCCZGCGTGGY (SEQ ID NO: 1), wherein P is a pyrimidine derivative mimicking a C/T mix, X is LNA-dA, and Z is LNA-dT.
2. The process of claim 1 wherein said reverse primer is the sequence of SEQ ID NO: 2.
3. The process of claim 1 wherein said measuring comprises hybridizing a probe of SEQ ID NO: 3 to said amplification product.
4. The process of claim 3 wherein hybridizing said probe is under conditions suitable for a polymerase chain reaction; and further
detecting a first detection signal from said probe hybridized to said amplification product.
5. The process of claim 1 wherein said detecting diagnoses human rhinovirus infection.
6. The process of claim 1 further comprising comparing said first detection signal to a second detection signal, wherein said second detection signal results from detection of a second amplification product produced from a sequence of a virus selected from the group comprising human enterovirus, polio virus, respiratory syncytial virus, human metapneumovirus, human parainfluenza viruses 1-4, adenovirus, coronaviruses 229E and OC43, influenza viruses A and B, and human bocavirus, and the hybridization of a probe complementary to a sequence from one or more viruses of said group.
7. The process of claim 1 further comprising comparing said first detection signal to a second detection signal, wherein said second detection signal results from detection of a second amplification product produced from a sequence of human rhinovirus using a forward primer homologous to a region within 356-563 of human rhinovirus, and a reverse primer homologous to a region within 356-563 of human rhinovirus, and the hybridization of a probe complementary to a sequence from human rhinovirus.
8. The process of claim 7 wherein said second detection signal is generated in parallel with said first detection signal.
9. The process of claim 7 , wherein said second amplification product is generated by PCR amplification of a HRV target sequence.
10. The process of claim 1 wherein said detecting is by real-time RT-PCR.
11. A kit for detecting human rhinovirus infection comprising: a first forward primer with sequence that is the isolated nucleotide sequence 5′-CPXGCCZGCGTGGY (SEQ ID NO: 1), wherein P is a pyrimidine derivative, X is LNA-dA, and Z is LNA-dT, and a first reverse primer with SEQ ID NO: 2; and
a probe.
12. The kit of claim 11 wherein said probe has the sequence SEQ ID NO: 3.
13. An isolated oligonucleotide of sequence comprising 5′-CPXGCCZGCGTGGY (SEQ ID NO: 1), wherein P is a pyrimidine derivative-mimicking a C/T mix, X is LNA-dA, and Z is LNA-dT.