Selective detection of <i>Haemophilus influenzae</i>

摘要

A process for detecting Haemophilus influenzae nucleic acid in a sample includes producing an amplification product by amplifying a Haemophilus influenzae nucleotide sequence and measuring the amplification product to detect Haemophilus influenzae in the sample. Some embodiments allow direct serotype determination in a single step assay. Also provided are reagents and methods for detecting and distinguishing Haemophilus influenzae from other infectious agents. A kit is provided for detecting and quantifying Haemophilus influenzae in a sample.

主权项

1. A process of detecting and distinguishing a serotype of Haemophilus influenzae in a sample comprising:
detecting the presence of the Haemophilus influenza protein D (hpd) gene in said sample or a portion thereof by a real-time polymerase chain reaction; producing an amplification product in a multiplex reaction by: contacting the sample or a second portion thereof with a forward primer consisting of SEQ ID NO: 1 that hybridizes to a Haemophilus influenzae serotype a capsule synthesis B (acsB) gene, and a reverse primer consisting of SEQ ID NO: 2 that hybridizes to a second region within said Haemophilus influenzae serotype a capsule synthesis B (acsB) gene under conditions suitable for a polymerase chain reaction; contacting the sample or the second portion thereof with a forward primer consisting of SEQ ID NO: 4 that hybridizes to a first region within a Haemophilus influenzae serotype b capsule synthesis B (bcsB) gene, and a reverse primer consisting of SEQ ID NO: 5 that hybridizes to a second region within said Haemophilus influenzae serotype b capsule synthesis B (bcsB) gene, under conditions suitable for a polymerase chain reaction; contacting the sample or the second portion thereof with a forward primer consisting of SEQ ID NO: 7 that hybridizes to a first region within a Haemophilus influenzae serotype c capsule synthesis D (ccsD) gene, and a reverse primer consisting of SEQ ID NO: 8 that hybridizes to a second region within said Haemophilus influenzae serotype c capsule synthesis D (ccsD) gene, under conditions suitable for a polymerase chain reaction; contacting the sample or the second portion thereof with a forward primer consisting of SEQ ID NO: 10 that hybridizes to a first region within a Haemophilus influenzae serotype d capsule synthesis E (dcsE) gene, and a reverse primer consisting of SEQ ID NO: 11 that hybridizes to a second region within said Haemophilus influenzae serotype d capsule synthesis E (dcsE) gene, under conditions suitable for a polymerase chain reaction; contacting the sample or the second portion thereof with a forward primer consisting of SEQ ID NO: 13 that hybridizes to a first region within a Haemophilus influenzae serotype e capsule synthesis H (ecsH) gene, and a reverse primer consisting of SEQ ID NO: 14 that hybridizes to a second region within said Haemophilus influenzae serotype e capsule synthesis H (ecsH) gene, under conditions suitable for a polymerase chain reaction; and contacting the sample or the second portion thereof with a forward primer consisting of SEQ ID NO: 16 that hybridizes to a first region within a Haemophilus influenzae serotype f export D (bexD) gene, and a reverse primer consisting of SEQ ID NO: 17 that hybridizes to a second region within said Haemophilus influenzae serotype f export D (bexD)gene, under conditions suitable for a polymerase chain reaction; wherein said step of producing is by subjecting the sample to cycling in a real-time PCR reaction in the presence of a probe comprising a fluorescent label; and detecting the presence of one or more Haemophilus influenzae serotypes in the sample by presence of said Haemophilus influenzae hpd gene and said amplification product.