Selective detection of Haemophilus influenzae
Inventors
Thomas, Jennifer • Wang, Xin • Hatcher, Cynthia • Anderson, Raydel • Theodore, Mary Jordan • Mayer, Leonard W.
Assignees
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Publication Number
US-10669591-B2
Publication Date
2020-06-02
Expiration Date
2032-01-26
Abstract
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.
Core Innovation
The invention provides processes and materials for the selective detection of Haemophilus influenzae (Hi) nucleic acid in a sample. The inventive process illustratively includes producing an amplification product by amplifying a Hi nucleotide sequence using forward and reverse primers that hybridize to specific regions within genes such as hpd, acsB, bcsB, ccsD, dcsE, ecsH, or Hif bexD. The amplification product is detected to identify the presence or absence of Hi in the sample, and optionally, a probe is used to measure this detection. Detection can inform diagnosis or treatment of Hi infection in a subject.
The process also allows for direct serotype determination in a single step assay by producing multiple amplification products using a plurality of primer sets capable of hybridizing to serotype-specific nucleotide sequences within the acsB, bcsB, ccsD, dcsE, ecsH, or Hif bexD genes. The invention further provides kits comprising primers and optionally non-degenerate probes for detecting and distinguishing Hi serotypes, enabling both detection and quantification in clinical, laboratory, or field settings.
The problem solved by the invention arises from the limitations of current methods for Hi detection and serotyping such as slide agglutination serotyping (SAST) and polymerase chain reaction (PCR). Conventional methods face challenges including subjective interpretation of SAST results and difficulty in detecting serotype replacement or vaccine failure. Additionally, non-typeable Hi has become the most prevalent cause of Hi-related infections due to widespread vaccination against serotype b. Therefore, there is a need for compositions and methods that improve the sensitivity, specificity, and rapidity of Hi detection and serotyping.
Claims Coverage
The claims define two primary inventive features related to detecting Haemophilus influenzae in a sample and kits for such detection.
Detection of Haemophilus influenzae hpd gene by PCR
A process of determining presence or absence of Haemophilus influenzae by combining a sample with a first forward primer comprising SEQ ID NO: 22 and a first reverse primer comprising SEQ ID NO: 23 that hybridize to specific portions of the hpd gene, under PCR conditions to produce an amplification product detectable in the sample.
Detection of additional Hi serotype-specific genes by PCR
Amplifying and detecting a second amplification product from a second Haemophilus influenzae nucleotide sequence within genes acsB, bcsB, ccsD, dcsE, ecsH, or Hif bexD using respective primers (selected from SEQ ID NOs: 1, 4, 7, 10, 13, 16 for forward and SEQ ID NOs: 2, 5, 8, 11, 14, 17 for reverse primers) to determine presence or absence of Hi in the sample.
Use of detectable probes in detection
Detecting amplification products by use of probes comprising sequences from SEQ ID NOs: 3, 6, 9, 12, 15, 18 or 24, including fluorescent labels and quenchers, enabling real-time or other detection modalities.
Kits for detecting Haemophilus influenzae infection
Kits comprising a first forward primer (SEQ ID NO: 22), a first reverse primer (SEQ ID NO: 23), and a first probe (SEQ ID NO: 24) with a detectable label capable of hybridizing to PCR amplification products, optionally including second sets of primers and probes for additional targets from acsB, bcsB, ccsD, dcsE, ecsH, or Hif bexD genes.
The claims cover sensitive and specific PCR-based processes and kits for detecting Haemophilus influenzae in samples by amplification of the hpd gene and optionally other serotype-specific genes, using designated primers and probes with detectable labels for identification and serotyping.
Stated Advantages
The invention provides rapid and sensitive detection of Haemophilus influenzae in biological samples.
It enables direct serotype determination in a single-step assay, improving diagnostics.
The methods and compositions improve specificity, distinguishing Haemophilus influenzae from other infectious agents without cross-reactivity.
The multiplex approach enhances confidence in Hi quantification and serotype identification by simultaneous or sequential detection of multiple targets.
Real-time PCR and mass spectrometry detection methods allow for quantitative, sensitive, and multiplexed analysis.
Documented Applications
Detection of Haemophilus influenzae in clinical, laboratory, or field samples, including human and animal biological specimens.
Diagnosis and monitoring of Haemophilus influenzae infections in subjects to inform treatment decisions.
Identification and serotyping of Haemophilus influenzae strains in surveillance, including monitoring for serotype replacement or vaccine failure.
Use of diagnostic kits comprising primers and probes for rapid and specific PCR-based detection and quantification of Haemophilus influenzae in suspected infection cases.
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