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-10683558-B2
Publication Date
2020-06-16
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 relates to processes and compositions for the selective detection of Haemophilus influenzae (Hi) nucleic acid in samples, particularly biological or environmental samples. The process involves producing an amplification product by amplifying a Hi nucleotide sequence using specific forward and reverse primers targeting genes such as hpd, acsB, bcsB, ccsD, dcsE, ecsH, or Hif bexD by polymerase chain reaction (PCR), and detecting the amplification product to identify the presence of Hi. The invention also provides reagents, probes, primer sets, and kits for both detecting and distinguishing Haemophilus influenzae from other infectious agents, including the ability for direct serotype determination in a single-step assay.
The problem addressed by the invention stems from the limitations of previous methods for identifying Hi and its serotypes, which include subjective interpretations of slide agglutination serotyping (SAST) results and variability in sensitivity and specificity of available serotyping reagents. The prevalence of non-typeable Hi infections, especially following vaccination programs targeting common serotype b (Hib), emphasizes the need for improved detection and serotyping methods. The invention aims to provide rapid, sensitive, specific, and robust molecular techniques for detection and serotyping, overcoming these previous shortcomings.
The invention also encompasses multiplex approaches that allow simultaneous detection and serotyping by using primers and probes corresponding to capsule biosynthesis or export genes specific to each serotype, along with pan-detection targeting conserved genes such as the hpd gene encoding protein D. The methods support different detection platforms including real-time PCR and mass spectrometry and include using nucleic acid calibrators or controls for quantification and increased accuracy. Diagnostic kits incorporating the described primers, probes and reagents are provided for clinical, laboratory, or field settings to facilitate diagnosis, epidemiological surveillance, and treatment guidance.
Claims Coverage
The patent claims focus on two main inventive features: molecular detection of Haemophilus influenzae by amplifying specific nucleotide sequences in the hpd gene and multiplex serotype identification through amplification of various capsule biosynthesis genes.
Process for detecting Haemophilus influenzae using hpd gene primers and probe
The invention features a process producing an amplification product by PCR using a first forward primer (SEQ ID NO: 22) and a first reverse primer (SEQ ID NO: 23) hybridizing to distinct regions within the hpd gene of Haemophilus influenzae and detecting the amplification product using a probe comprising SEQ ID NO: 24. Detection may involve fluorescent labeling and quenching, and detection signals can be compared to control samples or nucleic acid calibrators for increased fidelity.
Process for distinguishing Haemophilus influenzae serotypes via multiplex gene-specific amplification
The process includes producing a second amplification product by amplifying nucleotide sequences in genes specifically associated with Haemophilus influenzae serotypes, such as acsB, bcsB, ccsD, dcsE, ecsH, or Hif bexD using corresponding forward primers (e.g., SEQ ID NOs: 1, 4, 7, 10, 13, 16) and reverse primers (e.g., SEQ ID NOs: 2, 5, 8, 11, 14, 17), followed by detection of the amplification products, optionally using probes (SEQ ID NOs: 3, 6, 9, 12, 15, 18), allowing identification of the Haemophilus influenzae serotype present in the sample.
The claims cover processes for detecting Haemophilus influenzae by amplifying the hpd gene sequence with specific primers and probes, and for distinguishing its serotypes by multiplex PCR amplification of capsule biosynthesis/export genes with corresponding primers and probes. Detection strategies include fluorescent probes and comparative signal analysis with controls or calibrators, enabling rapid, sensitive, and specific identification and serotyping.
Stated Advantages
High specificity and sensitivity for detecting Haemophilus influenzae including all six serotypes and non-typeable strains.
Ability to rapidly and directly detect Haemophilus influenzae presence and serotype in single-step or multiplex assays.
Improved accuracy and objectivity over traditional serotyping methods such as slide agglutination, reducing variability and subjectivity.
Applicability to various detection platforms including real-time PCR and mass spectrometry enabling quantitative and multiplexed analyses.
Use of nucleic acid calibrators and controls enhances quantification accuracy and assay reliability.
Documented Applications
Diagnosis of Haemophilus influenzae infections in human clinical specimens such as cerebrospinal fluid and other biological samples.
Monitoring and surveillance of Haemophilus influenzae serotype prevalence and replacement, helping to detect vaccine failure or changes in pathogenic serotypes.
Multiplex detection and serotyping for epidemiological studies in hospital, laboratory, or field settings.
Quantification of bacterial load in samples to inform diagnosis and treatment decisions.
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