Rapid diagnostic kit using phage receptor binding proteins in lateral flow assay systems for bacterial identification

Abstract

Relates to a test method and test kit for the identification of the type, species or strain of bacteria contained in a sample, where the capture probe contains a bacteriophage receptor binding protein specific for the bacteria to be tested. The receptor binding protein can be labelled in conjugated form with gold nanoparticles.

Description

[0001] RAPID DIAGNOSTIC KIT USING PHAGE RECEPTOR BINDING PROTEINS IN LATERAL FLOW ASSAY SYSTEMS FOR BACTERIAL IDENTIFICATION

[0002] Technical Field

[0003] Relates to a test method and diagnostic kit for the identification of bacteria using bacteriophage receptor binding protein and lateral flow assay (LFA) technique.

[0004] State of The Art

[0005] Molecular methods for bacterial diagnosis are based on the detection of the specific DNA / RNA region of target bacteria, and different types of PCR such as multiplex polymerase chain reaction (mPCR), real-time PCR (RT-PCR), nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP) are used to study pathogenic bacteria.

[0006] Next-generation sequencing (NGS) is used in modern laboratories as an alternative to molecular methods. The major advantage of sequencing is the ability to detect even the most atypical microorganisms from a clinical sample, which can be used in sepsis or meningitis, but also in the diagnosis of tuberculosis.

[0007] Another method that is used for the identification of bacterial pathogens is the MALDI-TOF MS technique. Identification of the micro-organism by MALDI-TOF is based on comparison of the species-specific mass peaks with patterns in the database. This unique protein profile is referred to as a molecular "fingerprint".

[0008] The ELISA method, which is based on the interaction between antigen (Ag) and antibody (Ab), can also be used to detect bacteria in specimens.

[0009] These techniques have critical limitations in the detection of bacteria. There is therefore a need for methods based on biosensors that are easy to use, sensitive, specific, cost-effective, provide rapid, reproducible responses and offer numerous advantages, including easy on-site detection and portability.

[0010] Although many types of sensors for detecting bacteria have been described in the literature, these sensor systems are often complex, require technical skills and expertise, or consist of expensive equipment. A biosensor is defined as a device or system that detects a biochemical or physiological change through a probe, usually a biological component, and an electronic transducer that converts the biochemical signal into an audible electrical signal. A transducer is a device capable of amplifying the smallest biological response to a detectable signal that can be measured by various means, such as electrochemically, optically, acoustically, mechanically, calorimetrically or electronically, and then correlated with analyte concentration. Infection with a resistant isolate requires immediate on-site identification using rapid, economical and user-friendly methods. In this context, the lateral flow assay (LFA) technique, also known as rapid diagnostic test (RDT), has proven valuable in the detection and identification of antibiotic-resistant isolates.

[0011] One of the reasons why LFAs are so popular today is that they are paper-based. Paper-based materials are characterised by low cost, support sustainable production, porous matrices, suitability for device-free microfluidic systems and enable the design and strategy of modifiable biointeractive elements.

[0012] LFA is defined as a paper-based platform for the detection and quantification of analytes in complex mixtures, where the sample is placed in a test device and results are displayed within 5-30 minutes. Depending on the recognition elements used, LFA can be classified as antigenantibody interaction (Lateral Flow ImmunoAssay: LFIA) or DNA-DNA hybridisation (Nucleic Acid Lateral Flow ImmunoAssay: NALFIA or Nucleic Acid Lateral Flow: NAFL). The principle of LFA is based on the movement of the liquid specimen containing the analyte of interest through a series of polymeric strips to which molecules capable of interacting with the analyte are added by capillary action. Typically, LFA consists of overlapping membranes mounted on a backing board for improved stability and handling.

[0013] For a typical test on the LFA platform, the specimen is applied to an adsorbent specimen pad impregnated with buffer salts and surfactants to make it suitable for interaction with the detection system. The specimen pad ensures that the analyte in the specimen can bind to the capture reagents and membrane. The processed specimen passes through a conjugate release pad containing antibodies specific for the target analyte and conjugated to colored or fluorescent particles. The specimen passes along the strip with the conjugated antibody bound to the target analyte into the detection zone. This zone is a porous membrane, usually composed of nitrocellulose, on which specific biological components (usually antibodies or antigens) are immobilised in strips. Their role is to react with the analyte bound to the conjugated antibody. Detection of the sample analyte results in an appropriate response on the test line, while a response on the control line indicates appropriate fluid flow across the strip.

[0014] The reading, represented by lines of varying intensity, can be assessed by eye or with a specialised reader. To test multiple analytes simultaneously under the same conditions, additional antibody test lines specific for different analytes can be immobilised in an array format. On the other hand, multiple test lines loaded with the same antibody can be used for semi-quantitative analysis. The principle of this "lateral flow assay" test is based on the stepwise capture of colorimetric conjugate-antigen complexes by the immobilised antibody in each successive line, where the number of lines appearing on the strip is directly proportional to the concentration of the analyte. The liquid flows through the device due to the capillary force of the strip material and to sustain this movement, an absorbent pad is attached to the end of the strip. The role of the absorbent pad is to wick away excess reagents and prevent fluid reflux.

[0015] Literature examples are available for the determination of bacteria in LFA systems. And these examples often use antibodies as capture probes. The antibodies used are quite expensive and alternative methods are used due to both configuration problems and antibody degradation.

[0016] To date, numerous agents such as antimicrobial peptides, aptamers and antibiotics have been used to identify pathogenic bacteria on the LFA platform. However, antimicrobial peptides and antibiotics are not specific enough to identify specific target pathogens.

[0017] A review of the literature did not identify any studies using Receptor Binding Proteins (RBPs) as biosensors in LFA.

[0018] RBPs are very stable proteins with high resistance to proteases and detergents, as shown by studies in the literature. Overall, the high stability, specificity and ease of recombinant expression have made RBPs excellent alternatives to antibodies and ideal tools for the development of new diagnostic technologies. In addition, the ability of phages to be present in any environment in which their hosts can be found suggests that phage proteins may also have stable behavior in physiological fluids.

[0019] The interaction between RBPs on the phage and ligands / receptors on the bacterial (host) surface (lipopolysaccharides, outer membrane proteins, pili and flagella in Gram-negative bacteria; teichoic acid and lipoteichoic acid moieties in Gram-positive bacteria) is highly specific, and based on this binding, RBPs can be produced recombinantly or commercially.

[0020] Bacterio(phages) are the most widespread biological system in nature with approximately 1030particles. It creates its own life cycle by specifically infecting bacteria. Bacteriophages are characterised by their affinity for specific bacteria, which results from their ability to specifically recognise molecules present on the surface of host bacteria.

[0021] There are several methods that use bacteriophages to identify bacteria. EP3332022B1 and US2008 / 241819 disclose a method in which A1-labelled bacteriophage is used and unbound labelled bacteriophage is identified using a detection system comprising a photoacoustic cell.

[0022] US2001 / 006783 A1 discloses contacting a bacteriophage containing a dye or fluorochrome- labelled nucleic acid with a host bacterium and identifying the bacterium containing the dye or fluorochrome-labelled nucleic acid by optical detection means. Brief Description of the Invention

[0023] Bacteriophages bind to specific receptors on the surface of bacteria, so a bacteriophage can only identify bacteria that carry receptors to which it can bind.

[0024] The main objectives of the invention are to identify pathogens in samples from different sources with nanoparticle-labelled or unlabelled RBP; to provide on-site diagnosis with a portable, nonspecialised and highly specific RBP-based LFA biosensor; and to prevent disseminated infections by rapid identification of the infectious agent and effective isolation, and to avoid unnecessary broad-spectrum drug loading by using pathogen-specific drugs.

[0025] A lateral flow assay (LFA) rapid diagnostic kit has been developed to use host-sensitive phages in the search for diagnostic solutions to prevent the increasing loss of life and economic damage caused by resistant pathogens. In the invention, the principle of identification based on RBPs is used as the identifying molecule in the LFA system instead of the identification based on antibodies.

[0026] The aim of this invention is to develop a lateral flow test strip using bacteriophage RBP as a capture probe. The low cost and disposability of the developed test strip is an important advantage.

[0027] Detailed Description of the Invention

[0028] The invention is a test method for identifying the type, species or strain of bacteria contained in a specimen, characterised in that the capture probe (4) contains RBP. The RBP used can be labelled or unlabelled when conjugated to gold nanoparticles.

[0029] The invention further relates to a diagnostic kit for carrying out a test method for identifying the type, species or strain of bacteria contained in a specimen. The unique feature of the diagnostic kit is the use of a RBP that allows the bacteriophage to bind to its host for bacterial identification, and the use of a cobalt (Co-NTA) or nickel (Ni-NTA) metal chelated resin system instead of antibody capture, which is very economical and simple. A suitable cobalt (Co-NTA) or nickel (Ni-NTA) metal chelated resin system is commercially available (ThermoFisher). In addition, the RBP contained in the capture probe can be conjugated and labelled with gold nanoparticles.

[0030] For the purposes of the patent, the term "bacteria" should be understood as the host bacterium to which the bacteriophage RBPs bind.

[0031] The target microorganism specific RBP is used as the diagnostic element. For example, rGp144 is used to diagnose Staphylococcus aureus. The test method uses a positive sample containing the same type, species or strain of bacteria as the type, species or strain of bacteria to be detected and a specific phage RBP that can bind to the antigen contained in the type, species or strain of bacteria to be detected.

[0032] In the developed invention, the lateral flow platform comprises Nitrocellulose membrane (1), Support card (2), Test strip (3), Capture probe containing receptor binding protein (RBP) (4), Control strip (5), Metal chelated resin probe (6), Recognition probe (7), Conjugate pad (8), Sample and buffer application site on the pad (9) and Absorbent pad (10) parts (Figure 1).

[0033] Gold nanoparticles to be used as labels for the preparation of the platform / test strip to be used in the lateral flow assay can be synthesised or commercially available. Suitable solutions or buffers, which may contain various chemicals such as electrolytes, surfactants, etc., are used as mobile phase liquids in the analysis.

[0034] Testing can be performed in the following steps (Figure 2) using the developed diagnostic kit; a) Apply the specimen (12) and positive sample (11) to the pad (9), b) Place the diagnostic kit in the appropriate liquid medium and advance the specimen (12) and positive sample (11) on the pad until they come into contact with the recognition probe (7), c) The specific antigen in the positive sample (11) and, if present, in the specimen (12) binds to the recognition probe (7) and the conjugate formed moves towards the test strip (3), d) Contact of the conjugate with the RBP-containing capture probe (4) on the test strip and binding of the conjugate and RBP to form the conjugate-RBP compound-relevant phage host bacterium (e.g. S. aureus for rGp144) (13), e) Movement of the conjugate-RBP compound towards the control strip (5) and formation of conjugate-RBP compound - relevant phage host bacterium (e.g. S. aureus for rGp144) (13) for the positive control, or, if conjugate-RBP compound - relevant phage host bacterium (e.g. S. aureus for rGp144) (13) is not formed for the sample, binding of the RBP-unbound conjugate with the metal chelated resin.

[0035] If the specimen and the positive sample contain the same antigen, the conjugate-RBP compound (13) is formed for the positive control and the specimen and does not bind to the metal chelated resin in step e).

[0036] If the specimen and the positive sample do not contain the same antigen, the conjugate-RBP compound (13) is formed for the positive control, but the conjugate-RBP compound (13) is not formed for the specimen, and the conjugate-metal chelated resin compound (15), which is not bound by RBP, is formed in step (e). Accordingly, if the metal chelated resin is not conjugated in step e), the bacteria in the tested sample are the same as the bacteria in the positive sample. Otherwise, if the metal chelated resin is conjugated in step e), it is concluded that the bacteria in the tested sample are not the same as the bacteria in the positive sample.

[0037] The diagnostic kit can be packaged appropriately. The package may contain at least 20 RBPs suitable for the bacteria to be tested, metal chelated resin, and liquid as mobile phase. The name of the bacteria to be tested and instructions for use are printed on the package.

[0038] Description of figures:

[0039] Figure 1 . Schematic view of the bacterial diagnostic kit

[0040] Figure 2. Schematic view of the bacterial diagnostic process

[0041] Description of references:

[0042] 1- Nitrocellulose membrane

[0043] 2- Support card

[0044] 3- Test strip

[0045] 4- Capture probe containing receptor binding proteins (RBP)

[0046] 5- Control strip

[0047] 6- Metal chelated resin probe

[0048] 7- Recognition probe labelled or unlabelled with colloidal gold (e.g. AuNP modified with rGp144)

[0049] 8- Conjugate pad

[0050] 9- Pad containing sample and buffer application site

[0051] 10- Absorbent pad

[0052] 11- Positive sample

[0053] 12- Target antigen (host bacteria of the relevant phage (e.g. S. aureus for rGp144))

[0054] 13- Specific antigen with gold labelled capture probe conjugate

[0055] 14- Sandwich formation captured with capture probe

[0056] 15- Unbound labelled conjugate

[0057] For the evaluation of the developed LFA test kit in clinical samples, diagnosed samples from blood culture and nasal / throat swab samples from various clinics will be used in the Medical Microbiology Laboratory. In addition to its suitability for clinical use, it is thought to find applications in sectors that are important for environmental and public health, such as food, chemical, pharmaceutical and environmental analysis.

[0058] Additional studies of the validated LFA kit will be performed on blood and throat-nasal swab clinical samples diagnosed with S. aureus and non- S. aureus . At this stage, the same samples will be evaluated with a commercially available S. aureus LFA diagnostic kit and the results will be compared with the LFA kit developed as a result of the invention.

[0059] The main advantage of the paper-based systems produced by the invention is the low cost and the ability to make surface modifications specific to the target molecule. The invention is specific to a single analyte and is primarily targeted for use in the healthcare sector, enabling rapid analysis in areas such as clinical laboratories and intensive care units. Further studies will enable the preparation of specific diagnostic kits for various bacteria and kits that enable bacterial quantification, and it is envisaged that further patents will be filed for such developments of the invention.

[0060] The present invention provides a bacterial identification kit that enables the rapid and accurate determination of bacteria present in a test sample by a simple procedure. The present invention can therefore be used for sanitary control, process control and quality control in healthcare facilities, the food industry, the pharmaceutical industry and the like.

Claims

CLAIMS1 . A test method for identifying the type, species or strain of bacteria contained in a specimen, characterized in that it contains a bacteriophage receptor binding protein and uses a cobalt or nickel (Ni-NTA) metal chelated resin probe.

2. The test method of claim 1 comprising a specific phage receptor binding protein capable of binding to an antigen contained in the type, species or strain of bacteria to be detected.

3. The test method of claim 1 , wherein the bacteriophage receptor binding protein specific for the bacteria to be tested is labelled conjugated with gold nanoparticles.

4. The test method of claim 1 , wherein a positive sample containing the same bacterial type, species or strain as the bacterial type, species or strain to be detected is used.

5. The test method of claim 1 , wherein the lateral flow platform is used for the detection of bacteria.

6. The test method of claim 1 , comprising the following steps; a) Apply the specimen (12) and positive sample (11) to the pad (9), b) Place the diagnostic kit in the appropriate liquid medium and advance the specimen (12) and positive sample (11) on the pad until they come into contact with the recognition probe (7), c) The specific antigen in the positive sample (11) and, if present, in the specimen (12) binds to the recognition probe (7) and the conjugate formed moves towards the test strip (3), d) Contact of the conjugate with the RBP-containing capture probe (4) on the test strip and binding of the conjugate and RBP to form the conjugate-RBP compound (13), e) Movement of the conjugate-RBP compound towards the control strip (5) and formation of conjugate-RBP compound (13) for the positive control or, if conjugate-RBP compound (13) is not formed for the sample, the binding of the cobalt (Co-NTA) or nickel (Ni-NTA) metal chelated resin probe with the RBP-unbound conjugate.

7. The test method according to claim 6, wherein if the specimen and the positive sample are identical, the conjugate-RBP compound (13) is formed for the positive control and the specimen, and does not bind to the cobalt (Co-NTA) or nickel (Ni-NTA) 25 metal chelated resin probe in step e).

8. The test method according to claim 6, wherein if the specimen and the positive sample are not identical, the conjugate-RBP compound (13) is formed for the positive control, but the conjugate-RBP compound (13) is not formed for the specimen and an unbound RBP conjugate-cobalt (Co-NTA) or nickel (Ni-NTA) metal chelated resin (15) is formed in step e).

9. A diagnostic kit for the identification of the type, species or strain of bacteria contained in a specimen, characterized in that it uses a bacteriophage receptor binding protein specific to the bacteria to be tested the lateral flow platform has Nitrocellulose membrane (1), Support card (2), Test strip (3), Capture probe containing receptor binding protein (RBP) (4), Control strip (5), Cobalt (Co-NTA) or nickel (Ni-NTA) metal chelated resin probe for capture probe (6), Recognition probe (7), Conjugate pad (8), Pad containing sample and buffer application site (9) and Absorbent pad (10) parts, and the receptor binding protein contained in the capture probe is conjugated with gold nanoparticles, either labelled or unlabelled.

10. The diagnostic kit according to claim 9, wherein the receptor binding protein contained in the capture probe is labelled in conjugated form with gold nanoparticles.

11. The diagnostic kit according to claim 9, wherein the receptor binding protein contained in the capture probe is not labelled.

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