Selective detection of Haemophilus influenzae
Inventors
Thomas, Jennifer Dolan • Wang, Xin • Hatcher, Cynthia • Anderson, Raydel • Theodore, Mary Jordan • Mayer, Leonard W.
Assignees
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Publication Number
US-9593385-B2
Publication Date
2017-03-14
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 samples, including biological or environmental samples. A key process involves producing an amplification product by amplifying a Hi nucleotide sequence using forward and reverse primers targeting genes such as hpd, acsB, bcsB, ccsD, dcsE, ecsH, or Hif bexD under conditions suitable for polymerase chain reaction (PCR). Detection of the amplification product indicates the presence of Hi in the sample. The invention also allows for direct serotype determination in a single-step assay by using multiple primer sets targeting serotype-specific genes to distinguish among different capsule types a-f or non-typeable (NT) strains.
The problem addressed by the invention arises from the limitations of prior methods to detect and serotype Hi. Existing methods like slide agglutination serotyping (SAST) suffer from subjectivity and difficulty in interpreting weak agglutination reactions or variability in reagent specificity and sensitivity. Additionally, with vaccination programs reducing diseases from certain serotypes (notably serotype b), non-typeable Hi strains have become more prevalent, necessitating improved tools for detection and serotyping for monitoring serotype replacement and vaccine failure. Therefore, there is a need for compositions and methods that provide accurate, rapid, sensitive, and specific detection and serotyping of Hi.
The invention provides reagents such as primers and probes, optionally labeled with fluorescent molecules, designed to amplify and detect Hi nucleic acid sequences with high specificity and sensitivity. The processes include real-time quantitative PCR assays, multiplexed to simultaneously detect multiple serotypes, and employ probes to generate a detectable signal correlating with the presence of Hi. Additionally, kits containing these reagents are provided. The invention also contemplates the use of controls and nucleic acid calibrators to ensure assay accuracy and enable quantification of Hi in samples.
Claims Coverage
The claims present a process comprising detecting Haemophilus influenzae and simultaneously determining its serotype using a multiplex real-time PCR assay with specific primers and probes.
Simultaneous multiplex detection and serotyping of Haemophilus influenzae
The process includes detecting the presence of the hpd gene by real-time PCR in the sample and producing amplification products for serotype-specific genes (acsB for serotype a, bcsB for serotype b, ccsD for serotype c, dcsE for serotype d, ecsH for serotype e, and bexD for serotype f) in a multiplex PCR reaction using specific forward and reverse primers under suitable conditions.
Use of fluorescence-labeled probes for detecting amplification products
The detection step uses probes comprising specific nucleotide sequences (SEQ ID NOs: 3, 6, 9, 12, 15, or 18) labeled with a fluorescent reporter to generate a detectable signal indicative of amplification.
Comparison to control amplification products for validation
The process includes comparing the detection signal from the sample to signals derived from amplification products of control samples or nucleic acid calibrators to validate presence and quantity of Haemophilus influenzae.
The claims cover a multiplex real-time PCR process that detects the conserved hpd gene and simultaneously identifies specific serotypes of Haemophilus influenzae in a sample using specific primers and fluorescent probes, with steps for validation using control samples or calibrators, providing a method for sensitive and specific diagnosis.
Stated Advantages
Provides rapid and specific detection and serotyping of Haemophilus influenzae in biological and environmental samples.
Improves sensitivity and specificity over prior molecular targets such as ompP2 and bexA.
Enables multiplex detection of multiple serotypes simultaneously, increasing confidence and efficiency.
Reduces subjectivity and interpretation issues associated with slide agglutination serotyping methods.
Facilitates quantification of bacterial load via real-time PCR and use of nucleic acid calibrators.
Documented Applications
Diagnosis of Haemophilus influenzae infection in human subjects, including clinical isolates and cerebrospinal fluid from suspected meningitis patients.
Epidemiologic surveillance to detect and monitor Haemophilus influenzae serotype distribution and vaccine failure.
Laboratory and field detection of Haemophilus influenzae in various biological and environmental fluid samples.
Use in diagnostic kits comprising primers, probes, and reagents for detection and quantification of Haemophilus influenzae nucleic acid sequences.
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