A primer probe combination for simultaneously detecting AMB, VZV and HSV-1 and a detection method, application and product thereof

Abstract

This invention, entitled "A Primer-Probe Combination for Simultaneous Detection of AMB, VZV, and HSV-1, and its Detection Method, Application, and Product," belongs to the field of molecular biology detection technology. The technical problem it aims to solve is that existing ocular infection detection technologies cannot simultaneously detect AMB, VZV, and HSV-1 in a single tube, or only cover the virus and miss AMB, or require multiple tubes, have unclear identification, insufficient sensitivity, and are difficult to adapt to small ocular samples, failing to meet the needs of rapid and accurate clinical diagnosis. The key technical solution is to provide specific primers and probes targeting the 18S rRNA gene of AMB, the ORF62 gene of VZV, and the UL30 gene of HSV-1, combined with internal quality control primers and probes, using four different fluorescent labels to establish a single-tube multiplex real-time fluorescent PCR detection method, and preparing a matching kit to achieve simultaneous detection and identification of the three pathogens. This invention features high sensitivity, specificity, and strong anti-interference ability.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, and relates to nucleic acid detection technology for ocular infectious pathogens. Specifically, it relates to a primer-probe combination for simultaneous detection of AMB, VZV and HSV-1, as well as its detection method, application and product. Background Technology

[0002] Eye infections (such as keratitis and retinitis) caused by Acanthamoeba (AMB), varicella-zoster virus (VZV), and herpes simplex virus-1 (HSV-1) are serious blinding eye diseases that pose a significant threat to patients' visual health. Among them, *Acanthamoeba castellanii* is the most common and predominant pathogenic type of *Acanthamoeba*, the primary cause of *Acanthamoeba keratitis*, and a focus of attention for ophthalmology and infectious disease departments; Human herpesvirus 3 (HHV-3), also known as varicella-zoster virus (VZV), causes ophthalmic infections that are the most common cranial nerve complications, collectively known as herpes zoster ophthalmicus (HZO). The virus lies dormant in the ophthalmic branch (V1) of the trigeminal nerve, and after activation, it can invade the eye and its adnexa, affecting multiple parts such as the cornea, iris, and retina. Mild cases cause conjunctivitis, while severe cases can lead to blindness; HSV-1 Herpes simplex virus (HSK) is the most common pathogen causing ocular herpes infections. The resulting lesions are collectively known as herpes simplex eye disease. It is centered on corneal infection (herpes simplex keratitis) and can also affect the eyelids, conjunctiva, uvea, retina, and other parts of the eye. Its core characteristics are recurrent attacks and persistent symptoms. It can cause permanent vision damage due to corneal scarring, neovascularization, etc., and is one of the common causes of blindness in clinical practice.

[0003] The clinical manifestations of eye infections caused by these three pathogens often overlap, all potentially presenting with symptoms such as redness, pain, photophobia, and decreased vision. However, their treatment regimens are drastically different: AMB infection requires hydrazine or biguanide anti-amoebic drugs, while VZV and HSV-1 infections require specific antiviral drugs (such as acyclovir and valacyclovir). Therefore, rapid and accurate early differential diagnosis is crucial for guiding precise clinical medication, avoiding mistreatment, and preserving vision.

[0004] However, existing diagnostic techniques for these three pathogens have significant limitations: Traditional methods, such as corneal scraping microscopy and culture, are the "gold standard" for AMB diagnosis, but they are time-consuming (days to weeks) and have low sensitivity; viral culture is even more cumbersome and time-consuming, making it unsuitable for rapid diagnosis; while single-pathogen PCR detection has high sensitivity, it can only detect one pathogen per reaction. For suspected patients, clinicians often need to submit multiple individual tests sequentially, leading to prolonged diagnostic cycles, increased testing costs, and large sample consumption, which can easily delay optimal treatment. Regarding existing multiplex PCR technology, there is currently a lack of commercially available and clinically validated primer-probe combinations that can efficiently, evenly, and specifically detect AMB, VZV, and HSV-1 simultaneously. Furthermore, the sample volume available for ocular infections is extremely limited (e.g., aqueous humor, vitreous fluid, corneal scrapings), further increasing the difficulty of detection. Currently, there are no triple detection products for AMB, VZV, and HSV-1 for ocular infections.

[0005] Real-time fluorescence PCR technology has become the mainstream technology for detecting ocular pathogens due to its advantages such as high sensitivity, strong specificity, and rapid detection. Multiplex real-time fluorescence PCR technology can simultaneously detect multiple pathogens in a single tube and a single reaction, effectively addressing the pain points of limited sample collection and high-efficiency testing requirements in ophthalmology clinical practice, and has become an important development direction in this field. Currently, several PCR detection technologies targeting ocular infectious pathogens have been developed and published, but significant shortcomings remain, failing to meet actual clinical needs.

[0006] Relevant patent documents retrieved:

[0007] Country of Origin: China, Publication Number: CN 112080586 A, Publication Date: December 15, 2020. This document discloses a solid-phase multiplex PCR detection kit and method for infectious eye pathogens. It uses solid-phase treated primers and probes combined with triplex PCR tubes to simultaneously detect eight herpesviruses. The solid-phase technology reduces primer dimer production, improves amplification efficiency, and saves eye sample volume. However, the detection targets only cover herpesviruses and do not include Acanthamoeba. It is necessary to detect multiple targets in triplex PCR tubes separately, which fails to achieve simultaneous identification of multiple targets in a single tube. The operation process is relatively cumbersome and cannot meet the detection needs of mixed infection of protozoa and viruses.

[0008] Country of Origin: China, Publication Number: CN 116555496 A, Publication Date: August 8, 2023. This document discloses a primer and probe set, detection method, and kit for multiplex PCR detection of common ophthalmic viruses. Employing multiplex PCR technology, it can simultaneously detect five viruses—adenovirus, cytomegalovirus, HSV-1, HSV-2, and VZV—in a single tube. The reaction system has strong anti-interference capabilities and can be directly tested using raw ocular samples without nucleic acid extraction. However, the detection range is limited to viral pathogens, excluding Acanthamoeba. Furthermore, HSV-1 and VZV share the ROX fluorescence channel, allowing only joint determination of positive results and failing to accurately distinguish between the two viruses. The detection sensitivity is 500 copies / mL, and its ability to detect low-concentration infected samples is limited.

[0009] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects:

[0010] 1. Incomplete target coverage, unable to meet the needs of detecting mixed infections of protozoa and viruses: CN 112080586 A only targets 8 types of herpesviruses, and CN 116555496 A only detects 5 types of viruses. Neither of them includes Acanthamoeba, an important protozoan causing ocular infections. However, mixed infections of AMB with VZV and HSV-1 are not uncommon in clinical practice, and current technology cannot achieve simultaneous detection of such mixed infections.

[0011] 2. The single-tube multi-target identification capability is insufficient. CN 112080586 A requires the use of triplet PCR tubes to detect different viruses separately, and does not achieve simultaneous identification in a single tube. In CN 116555496 A, HSV-1 and VZV share the same fluorescence channel, which can only jointly determine the positive result, and cannot accurately distinguish between the two pathogens, which may lead to insufficient drug targeting.

[0012] 3. Some technologies have limited sensitivity and are difficult to meet the needs of early diagnosis: CN 116555496 A has a detection sensitivity of 500 copies / mL, which may result in missed detection for low-concentration infection samples in the early clinical stage.

[0013] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles:

[0014] 1. HSV-1 and VZV both belong to the alpha herpesvirus family and have extremely high homology. They are both herpesviridae and have conserved genome structures. Therefore, non-specific cross-reactions of primers or probes are likely to occur, resulting in cross-amplification and false positives, which makes it difficult to select target gene sequences and design primers and probes in multiplex amplification systems.

[0015] 2. At the same time, HSV-1 and HSV-2 (herpes simplex virus type 2) belong to the same herpes simplex virus family, with about 50%-80% homology in their whole genome. The homology of conserved genes (such as gB, gH, DNA polymerase, etc.) exceeds 85%. Even in the highly variable and low homology regions of the gG gene, there are still continuous homologous fragments, which can easily cause non-specific binding of primers and probes and false positive results.

[0016] 3. Integrating multiple viruses of the same genus and high homology into the same detection product requires rigorous verification and evaluation of the specificity of the target gene sequence, primers and probes. At the same time, it is necessary to ensure the detection sensitivity to adapt to clinical specimens with small sample volumes, such as ophthalmic swabs, scrapings, and aqueous humor, which significantly increases the difficulty of product reaction system design. Summary of the Invention

[0017] The purpose of this invention is to provide:

[0018] A highly sensitive, specific, and compatible multiplex real-time fluorescent PCR primer-probe combination has been developed. This combination enables simultaneous detection and identification of AMB, VZV, and HSV-1 in a single tube and single reaction, thereby enabling the development of a rapid and reliable diagnostic kit to meet the urgent clinical need for rapid and accurate diagnosis of eye infections.

[0019] Terminology Explanation:

[0020] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0021] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0022] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0023] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0024] Multiplex Real-time PCR: The core detection technology of this invention refers to the simultaneous detection of three targets—Acanthamoeba (AMB), varicella-zoster virus (VZV), and herpes simplex virus type 1 (HSV-1)—in a single tube and single PCR reaction by using multiple sets of specific primers and probes combined with different fluorescent labels, thereby achieving simultaneous identification of multiple pathogens.

[0025] Primer-probe Set: The core detection component consists of specific primer pairs and probes targeting three targets and internal quality control. Primers are used to specifically amplify the target gene, and probes are used to capture real-time fluorescence signals to confirm the presence of the target.

[0026] Target gene: A specific gene fragment used for pathogen detection.

[0027] Internal Control (IC): A quality control system designed based on the Bacillus gyrB gene (helicase B subunit gene) to monitor the effectiveness of the entire process of nucleic acid extraction and PCR amplification and avoid false negative results.

[0028] Fluorescent Labeling System: This system uses a combination of non-interfering fluorescent groups and quenching groups to distinguish signals from multiple targets.

[0029] Ocular Sample: The clinical test sample applicable to this invention includes samples collected from ocular infection sites such as aqueous humor, corneal scrapings, conjunctival smears, and tears. The sample volume is small.

[0030] Endogenous interferents: Substances that may be present in eye samples that affect the PCR reaction, including hemoglobin, albumin, bilirubin, etc.

[0031] The technical solution of this invention:

[0032] On the one hand, the present invention provides a primer-probe combination for simultaneous detection of AMB, VZV and HSV-1, including specific primer pairs and probes for AMB, VZV and HSV-1 genes, as well as internal quality control primer pairs and probes.

[0033] The primer pair sequences of the AMB are shown in SEQ ID NO.1-2, and the probe sequence is shown in SEQ ID NO.7;

[0034] The primer pair sequences of the VZV are shown in SEQ ID NO.3-4, and the probe sequence is shown in SEQ ID NO.8;

[0035] The primer pair sequences of the HSV-1 are shown in SEQ ID NO.5-6, and the probe sequence is shown in SEQ ID NO.9;

[0036] The primer pair sequences for the internal quality control are shown in SEQ ID NO.10-11, and the probe sequences are shown in SEQ ID NO.12.

[0037] Specifically, the probes for AMB, VZV, HSV-1, and internal quality control are labeled with mutually non-interfering fluorescent groups, and each probe is labeled with a quencher suitable for the corresponding fluorescent group.

[0038] More specifically, the fluorescent group is selected from any four of FAM, ROX, CY5, VIC, HEX, and TAMRA; the quencher is selected from one or more of BHQ series quenchers and TAMRA.

[0039] Preferably, the probe corresponding to AMB is labeled with FAM fluorescent group and BHQ1 quencher, the probe corresponding to VZV is labeled with ROX fluorescent group and BHQ2 quencher, the probe corresponding to HSV-1 is labeled with CY5 fluorescent group and BHQ3 quencher, and the probe corresponding to internal quality control is labeled with VIC fluorescent group and BHQ1 quencher.

[0040] Specifically, the final concentrations of each primer and probe can be as follows: the final concentrations of the forward and reverse primers for AMB, VZV, and HSV-1 are all 0.2-0.28 μM; the final concentrations of the corresponding probes are all 0.1-0.14 μM; the final concentrations of the internal quality control forward primers are 0.14-0.18 μM, the final concentrations of the reverse primers are 0.1-0.14 μM, and the final concentrations of the probes are 0.06-0.10 μM.

[0041] Preferably, the final concentrations of each primer and probe are as follows: the final concentrations of the forward and reverse primers for AMB, VZV, and HSV-1 are all 0.24 μM; the final concentrations of the corresponding probes are all 0.12 μM; the final concentrations of the internal quality control forward primer are 0.16 μM, the final concentrations of the reverse primer are 0.12 μM, and the final concentrations of the probes are 0.08 μM.

[0042] In another aspect, the present invention provides a detection method for simultaneously detecting AMB, VZV, and HSV-1, comprising the following steps:

[0043] (1) Nucleic acid extraction from samples;

[0044] (2) Prepare a PCR reaction system containing the aforementioned primer and probe combination;

[0045] (3) PCR amplification and result determination.

[0046] Specifically, the sample mentioned in step (1) is an ocular sample, including aqueous humor, corneal scraping, conjunctival smear or tear fluid.

[0047] Specifically, the PCR amplification program in step (3) is as follows: incubation at 37℃ for 1-3 min, pre-denaturation at 94-96℃ for 25-35 s; followed by denaturation at 94-96℃ for 4-6 s, annealing and extension at 58-62℃ for 18-22 s, for a total of 38-42 cycles.

[0048] Preferably, the PCR amplification program in step (3) is as follows: incubation at 37°C for 2 min, pre-denaturation at 95°C for 30 s; followed by denaturation at 95°C for 5 s, annealing and extension at 60°C for 20 s, for a total of 40 cycles.

[0049] Specifically, the criteria for interpreting the results in step (3) are as follows: Validity of the test results: After the PCR reaction is completed, the Ct value of the VIC channel is read. If the Ct value of the VIC channel is ≤33, the test result is considered valid; otherwise, the test result is invalid and the experiment needs to be repeated. Validity of Acanthamoeba (AMB) test results: If the Ct value of the FAM channel is ≤38 under the premise that the test result is valid, the test result is considered positive for AMB; otherwise, the test result is not positive for AMB. Validity of varicella-zoster virus (VZV) test results: If the Ct value of the ROX channel is ≤37 under the premise that the test result is valid, the test result is considered positive for VZV; otherwise, the test result is not positive for VZV. Validity of herpes simplex virus type 1 (HSV-1) test results: If the Ct value of the ROX channel is ≤37 under the premise that the test result is valid, the test result is considered positive for HSV-1; otherwise, the test result is not positive for HSV-1.

[0050] In another aspect, the present invention provides a kit comprising the aforementioned primer-probe combination, PCR premix, internal quality control, positive control and negative control.

[0051] Specifically, the PCR premix contains dNTP / dUTP, Mg ions, and hot-start DNA polymerase; the positive control is a recombinant plasmid containing the conserved AMB gene, the conserved VZV gene, and the conserved HSV-1 gene; and the negative control is a blank plasmid without the target gene or nuclease-free water.

[0052] In another aspect, the present invention provides the application of the aforementioned primer-probe combination or kit in the preparation of products for the diagnosis of ocular infectious pathogens.

[0053] Specifically, the eye infections include, but are not limited to, keratitis, retinitis, uveitis, or conjunctivitis.

[0054] The present invention also provides the following technical solutions:

[0055] A diagnostic method for an eye infection includes the following steps:

[0056] (1) Obtain eye-derived samples from the subjects;

[0057] (2) The aforementioned detection methods were used to detect AMB, VZV and HSV-1 nucleic acids in the samples;

[0058] (3) Determine whether the subject is infected with AMB, VZV and / or HSV-1 based on the test results, and then diagnose the type of pathogen causing the eye infection: if AMB nucleic acid is positive, it is determined to be an eye infection caused by AMB infection; if VZV nucleic acid is positive, it is determined to be an eye infection caused by VZV infection; if HSV-1 nucleic acid is positive, it is determined to be an eye infection caused by HSV-1 infection; if two or three pathogen nucleic acids are positive, it is determined to be an eye infection caused by mixed infection; if none of them are detected, then eye infection caused by AMB, VZV and HSV-1 infection is excluded.

[0059] Specifically, the ocular samples include aqueous humor, corneal scrapings, conjunctival smears, or tears.

[0060] Specifically, the ocular infection includes keratitis, retinitis, uveitis, or conjunctivitis; the diagnosis also includes combining the subject's clinical symptoms, which include one or more of the following: eye pain, photophobia, tearing, decreased vision, conjunctival congestion, corneal opacity, and fundus exudation.

[0061] The present invention has at least the following beneficial effects:

[0062] (1) The technical solution of the present invention can identify three pathogens in a single reaction, which greatly shortens the diagnosis cycle, reduces the consumption of samples and reagents, and is suitable for the clinical characteristics of small sample volume of ocular infection.

[0063] (2) The technical solution of the present invention is designed to target conserved gene regions without cross-reaction; it can detect as few as 4 copies of the target, accurately identify low-load pathogens, and achieve early infection screening.

[0064] (3) The technical solution of the present invention can effectively eliminate experimental errors, avoid false negatives, and ensure the accuracy of test results.

[0065] (4) The technical solution of the present invention is resistant to common endogenous interference and is compatible with a variety of ocular clinical samples; the operation is standardized and the reaction conditions are uniform, making it easy to implement in routine laboratories.

[0066] (5) The technical solution of the present invention solves the problem of identifying mixed infections, guides targeted medication, reduces serious complications such as visual impairment, and lowers the blindness rate caused by eye infections, which has important clinical and social value. Attached Figure Description

[0067] Figure 1 This is a schematic diagram of the linear detection results of AMB 18S rRNA.

[0068] Figure 2 This is a schematic diagram of the linear detection results of VZV ORF62.

[0069] Figure 3 This is a schematic diagram of the HSV-1 UL30 linearity test results.

[0070] Figure 4 This is a schematic diagram of the internal quality control (IC) test results in a linear detection experiment.

[0071] Figure 5 This is a schematic diagram showing the detection limit concentration of AMB 18S rRNA.

[0072] Figure 6 This is a schematic diagram of the detection limit concentration of VZV ORF62.

[0073] Figure 7 This is a schematic diagram of the HSV-1 UL30 detection limit concentration test results.

[0074] Figure 8 This is a schematic diagram showing the detection results of a mixed sample containing low-concentration AMB, high-concentration VZV, and high-concentration HSV-1.

[0075] Figure 9 This is a schematic diagram showing the detection results of a mixed sample of medium-concentration AMB, low-concentration VZV, and high-concentration HSV-1.

[0076] Figure 10 This is a schematic diagram showing the detection results of a mixed sample containing high-concentration AMB, high-concentration VZV, and medium-concentration HSV-1.

[0077] Figure 11 This is a schematic diagram showing the detection results of a mixed sample containing high-concentration AMB, low-concentration VZV, and medium-concentration HSV-1.

[0078] Figure 12 This is a schematic diagram showing the detection results of a mixed sample containing low-concentration AMB, high-concentration VZV, and medium-concentration HSV-1. Detailed Implementation

[0079] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0080] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0081] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0082] Example 1: Primer and probe composition for the detection of Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1 nucleic acid.

[0083] Primers and probes were designed based on the 18S ribosomal RNA sequence U07401.1 in the genome nucleic acid sequence of Acanthamoeba castellanii CDC 0184 in GenBank, using the primer design software Primer Premier 5.

[0084] Based on the varicella-zoster virus (VZV) genome nucleic acid sequence NC_001348.1 in GenBank, the VZV ORF62 gene with high specificity and high sensitivity was selected, and primers and probes were designed using the primer design software Primer Premier 5.

[0085] Based on the sequence NC_001806.2 of herpes simplex virus type 1 (HSV-1) strain17 in GenBank, the UL30 gene with high specificity and high sensitivity was selected, and primers and probes were designed using the primer design software Primer Premier 5.

[0086] Based on the chromosome sequence CP166481.1 of Bacillus atrophaeus strain globigii in GenBank, the gyrB gene was selected, and internal quality control (IC) primers and probes were designed using the primer design software Primer Premier 5. At the same time, internal quality control (IC) plasmids were synthesized.

[0087] The specific methods for primer and probe design are as follows:

[0088] (1) Primers are designed in conserved sequence regions, and probes are designed between upstream and downstream primers;

[0089] (2) The fluorescent group of the AMB 18S rRNA probe is labeled with the universal FAM probe dye, and the quenching group is labeled with the universal BHQ1 probe quencher; the fluorescent group of the VZV ORF62 probe is labeled with the universal ROX probe dye, and the quenching group is labeled with the universal BHQ2 probe quencher; the fluorescent group of the HSV-1 UL30 probe is labeled with the universal CY5 probe dye, and the quenching group is labeled with the universal BHQ3 probe quencher; the fluorescent group of the internal quality control (IC) probe is labeled with the universal VIC probe dye, and the quenching group is labeled with the universal BHQ1 probe quencher.

[0090] The sequence information of the primers and probes is shown in Table 1.

[0091] Table 1 Primer and probe sequence information

[0092]

[0093] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; and "P" represents the probe.

[0094] Example 2: Kit and Usage Method for Detecting Acanthamoeba, Varicella-zoster Virus, and Herpes Simplex Virus Type 1 Nucleic Acid

[0095] 1. Reagent preparation (solution preparation area)

[0096] (1) First, remove all reagents from the refrigerator and allow them to equilibrate to room temperature.

[0097] (2) Preparation of primer and probe mixture: Prepare primer and probe mixture according to the composition in Table 2.

[0098] Table 2 Composition of primer-probe mixture

[0099]

[0100] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; and "P" represents the probe.

[0101] (3) Transfer the prepared primer-probe mixture to the extraction room and store it at 4℃ or -20℃ until the sample extraction process is completed.

[0102] 2. Sample extraction (extraction area)

[0103] (1) Nucleic acid extraction from the sample to be tested

[0104] Nucleic acid extraction, enrichment, and purification steps are performed using nucleic acid extraction or purification reagents. In this embodiment of the invention, the Magnetic Serum / Plasma DNA Maxi Kit (purchased from Tiangen Biotech, catalog number DP710) is used, and the nucleic acid enrichment method is the magnetic bead capture method.

[0105] (2) Prepare the PCR reaction solution according to the composition in Table 3, add it to a 1.5 mL centrifuge tube, vortex for a few seconds, centrifuge at 3000 rpm for a few seconds. The prepared PCR reaction solution can be used within 1 hour at 4℃ or within 4 hours at -20℃. The PCR premix contains dNTP / dUTP, Mg ions, DNA polymerase and other components required for PCR reaction. In this embodiment of the invention, 2x All-Powerful qPCR PreMix (purchased from Novizan Biotechnology, catalog number QN-222) is used.

[0106] Table 3 Composition of PCR reaction solution

[0107]

[0108] (3) Use a micropipette to dispense the prepared PCR reaction solution into PCR thin-walled reaction tubes in 31 μL portions.

[0109] (4) Use a micropipette to add 20 μL of the corresponding DNA extraction sample, negative control or positive control to each PCR thin-walled reaction tube, immediately close the tube cap tightly, mix well and then centrifuge briefly.

[0110] (5) Transfer the PCR thin-walled reaction tube containing the template to the PCR amplification area.

[0111] 3. PCR amplification (PCR amplification region)

[0112] (1) The PCR amplification program is set as follows:

[0113] Step 1: 37℃, 2 minutes;

[0114] Step 2: 95℃, 30s;

[0115] Step 3: 95℃, 5s; 60℃, 20s; 40 cycles; collect fluorescence signals at 60℃, 20s, using FAM, VIC, ROX and CY5 fluorescence channels.

[0116] (2) After the program is completed, take out the PCR thin-walled reaction tube and put it into the embossed bag. Seal the bag tightly and treat it as a source of contamination.

[0117] 4. Result Determination

[0118] Determining the validity of test results: After the PCR reaction is completed, read the Ct value of the VIC channel. If the Ct value of the VIC channel is ≤33, the test result is considered valid; otherwise, the test result is invalid and the experiment needs to be repeated.

[0119] Interpretation of Acanthamoeba (AMB) detection results: If the FAM channel Ct value is ≤38, the result is considered positive for AMB; otherwise, it is considered undetectable for AMB.

[0120] Interpretation of varicella-zoster virus (VZV) test results: If the ROX channel Ct value is ≤37, the test result is considered positive for VZV; otherwise, VZV is not detected.

[0121] Interpretation of HSV-1 test results: If the ROX channel Ct value is ≤37, the test result is considered positive for HSV-1; otherwise, it is considered undetectable for HSV-1.

[0122] Example 3: Linear range of nucleic acid detection kits for Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1.

[0123] The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0124] The samples were AMB 18S rRNA plasmid (GenBank sequence U07401.1), VZV ORF62 plasmid (GenBank sequences NC_001348.1, 105141..109133), and HSV-1 UL30 plasmid (GenBank sequences NC_001806.2, 62807..66553), synthesized by Shanghai Jereh Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently.

[0125] In this embodiment, each plasmid was serially diluted 10-fold using TE buffer to prepare test samples of different concentrations, with a final concentration ranging from 800 cpoies / mL to 4E8 cpoies / mL. The samples were tested using the kit described in Example 2, following the method described in Example 2, with four replicates for each sample.

[0126] A schematic diagram of the linear detection range results of AMB is shown below. Figure 1 As shown; a schematic diagram of the linear detection range of VZV is shown below. Figure 2 As shown; a schematic diagram of the linear detection range results for HSV-1 samples is shown below. Figure 3 As shown; a schematic diagram of the internal quality control (IC) test results is shown below. Figure 4 As shown.

[0127] The detection Ct values ​​for each sample were statistically analyzed, and the coefficient of variation (CV, %) was calculated. The results are as follows:

[0128] Table 4 Repeatability of Linear Sample Detection

[0129]

[0130] Continued from Table 4: Repeatability of Linear Sample Detection

[0131]

[0132] The results show that the kit for detecting Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1 nucleic acid of the present invention can detect samples at different concentrations. This indicates that the kit can achieve effective detection over a wide concentration range, meeting the detection needs of samples with different levels of infection. In terms of repeatability, the coefficients of variation for each concentration gradient of the three target pathogens are at low levels, meeting the repeatability standards for nucleic acid detection kits. This indicates that the kit's detection results are stable, reliable, and have good repeatability, effectively avoiding misjudgments caused by detection errors.

[0133] Example 4: Sensitivity of the nucleic acid detection kit for Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1

[0134] The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0135] The samples were AMB 18S rRNA plasmid, VZV ORF62 plasmid, and HSV-1 UL30 plasmid, synthesized by Shanghai Jereh Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently.

[0136] In this embodiment, each plasmid was diluted with TE buffer to prepare a test sample with a concentration of 400 copies / mL. 10 μL was used for detection, that is, the initial amount of each detection target was 4 copies. The sample was measured using the kit described in Example 2, and the method described in Example 2 was followed.

[0137] A schematic diagram of AMB sensitivity detection results is shown below. Figure 5 As shown; a schematic diagram of the sensitivity detection results of VZV is shown below. Figure 6 As shown; a schematic diagram of the sensitivity detection results of HSV-1 is shown below. Figure 7 As shown.

[0138] The detection Ct values ​​for each sensitivity sample (4 copies) were statistically analyzed, and the coefficient of variation (CV, %) was calculated. The results are as follows:

[0139] Table 5 Sensitivity Sample Detection Repeatability

[0140]

[0141] The results show that the kit for detecting Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1 nucleic acid of the present invention can detect samples with starting levels as low as single digit copy numbers. The kit effectively detects low starting levels of all three target pathogens, and the detection repeatability for each low starting level sample is good, with a low coefficient of variation, meeting the sensitivity-related detection standards for nucleic acid detection kits. This indicates that the kit has extremely high detection sensitivity, accurately capturing target nucleic acids in low-concentration infected samples, and can meet the clinical needs for detecting low-load samples such as latent infections and early infections.

[0142] Example 5: Detection of mixed infection with Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1.

[0143] The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0144] The samples were AMB 18S rRNA plasmid, VZV ORF62 plasmid, and HSV-1 UL30 plasmid, synthesized by Shanghai Jereh Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently.

[0145] In this embodiment, TE buffer was used to dilute each plasmid separately to prepare high-concentration (4E7 copies / mL), medium-concentration (4E3 copies / mL), and low-concentration (400 copies / mL) test samples. The samples of different targets were mixed in high, medium, or low concentrations. The mixed samples were then measured using the kit described in Example 2, following the method described in Example 2.

[0146] A schematic diagram of the detection results of mixed samples with medium concentration AMB, high concentration VZV and high concentration HSV-1 is shown below. Figure 8 As shown in the diagram; a schematic diagram of the detection results of a mixed sample of high-concentration AMB, low-concentration VZV, and high-concentration HSV-1 is shown in the diagram. Figure 9 As shown in the diagram; a schematic diagram of the detection results of a mixed sample of high-concentration AMB, high-concentration VZV, and medium-concentration HSV-1 is shown in the diagram. Figure 10 As shown in the diagram; a schematic diagram of the detection results of a mixed sample of high-concentration AMB, low-concentration VZV, and medium-concentration HSV-1 is shown in the diagram. Figure 11 As shown in the figure; a schematic diagram of the detection results of a mixed sample of medium-concentration AMB, high-concentration VZV, and medium-concentration HSV-1 is shown in the figure. Figure 12 As shown. The results indicate that the kit for detecting Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1 nucleic acid of the present invention can detect mixed infection samples with different viral loads.

[0147] Example 6: Interference Inhibition Experiment for Detection of Mixed Infections of Acanthamoeba, Varicella-Zoster Virus, and Herpes Simplex Virus Type 1

[0148] The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0149] The samples were AMB 18S rRNA plasmid, VZV ORF62 plasmid, and HSV-1 UL30 plasmid, synthesized by Shanghai Jereh Biotechnology Co., Ltd. Each plasmid was synthesized and quantified independently.

[0150] In this embodiment, each plasmid was diluted with TE buffer to prepare a test sample with a concentration of 400 copies / mL. 20 μL was used for detection, that is, the initial amount of each detection target was 8 copies. The sample was measured using the kit described in Example 2, and the method described in Example 2 was followed.

[0151] The control group contained no interfering substances and was a mixed sample near the detection limit. The interfering substance groups contained samples containing 2 mg / mL hemoglobin, 50 mg / mL albumin, or 200 μg / mL bilirubin, respectively, mixed with the three target plasmids to prepare the test samples. The kits described in Example 2 were used to analyze each sample, following the method described in Example 2. The Ct values ​​for each group are shown in Table 6. The results indicate that the kit for detecting Acanthamoeba, varicella-zoster virus, and herpes simplex virus type 1 nucleic acid of the present invention is resistant to common endogenous interfering substances.

[0152] Table 6. Suppression Test of Potential Interfering Substances

[0153]

[0154] Example 7: Detection experiment for ophthalmic samples from clinical patients

[0155] The samples were measured using the kit described in Example 2, following the method described in Example 2.

[0156] The samples were collected from the ophthalmology outpatient department of Suzhou Dushu Lake Hospital. The accuracy of the test results was evaluated using clinical tNGS test results as a control. The consistency of the test results for various pathogen infection samples is shown in the table below:

[0157] Table 7 Accuracy of Clinical Ophthalmology Sample Test Results

[0158]

[0159] Comparative Example 1: A kit containing primers and probes for different detection targets.

[0160] The only difference between Comparative Example 1 and Example 1 is that the primer and probe compositions for the test targets (AMB, VZV, and HSV-1) are different, while the primer and probe compositions for the internal quality control (IC) are the same. The primer and probe mixtures were prepared according to Table 8.

[0161] Table 8 Primer and probe sequence information

[0162]

[0163] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; and "P" represents the probe.

[0164] Comparative Example 2: Kits containing mixtures of different primer and probe concentrations

[0165] The only difference between Comparative Example 2 and Example 2 is that the primer and probe concentrations for the target molecules (AMB, VZV, and HSV-1) are different, while the primer and probe concentrations for internal quality control (IC) are the same. Primer and probe mixtures were prepared according to the concentrations in Table 9.

[0166] Table 9 Comparative Example 2: Primer and probe ratios at different concentrations

[0167]

[0168] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer; and "P" represents the probe.

[0169] Experimental Example 1

[0170] In Comparative Example 1, three plasmid samples diluted with TE buffer were mixed in equal proportions, resulting in a concentration of 1E4 copies / mL for the three plasmids. Test samples were prepared for this purpose. The test results for different primer-probe composition systems compared to the present invention are shown in Table 10.

[0171] Table 10 shows the test results for different primer and probe compositions in Comparative Example 1.

[0172]

[0173] Experiment Example 2

[0174] In Comparative Example 2, the three plasmid samples diluted with TE buffer were mixed. The concentrations of the three plasmids after mixing were AMB 1E5 copies / mL, VZV 1E4 copies / mL, and HSV-1 400 copies / mL, respectively, forming mixed target samples with different high and low concentrations.

[0175] The test results of the primer-probe reaction system with different concentrations are shown in Table 11:

[0176] Table 11. Test results of primer and probe reaction systems with different concentrations in Comparative Example 2.

[0177]

[0178] The test results show that the primer-probe reaction system with concentration 3 (in this invention) has the lowest Ct value for high-concentration target, and only the primer-probe reaction system with concentration 3 can stably measure the Ct value in the detection limit concentration test of target, with the highest detection efficiency. Therefore, concentration 3 is the preferred concentration.

[0179] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A primer probe combination for simultaneous detection of AMB, VZV and HSV-1, characterized in that, This includes specific primer pairs and probes for the AMB, VZV, and HSV-1 genes, as well as internal quality control primer pairs and probes. The primer pair sequences of the AMB are shown in SEQ ID NO.1-2, and the probe sequence is shown in SEQ ID NO.7; The primer pair sequences of the VZV are shown in SEQ ID NO.3-4, and the probe sequence is shown in SEQ ID NO.8; The primer pair sequences of the HSV-1 are shown in SEQ ID NO.5-6, and the probe sequence is shown in SEQ ID NO.9; The primer pair sequences for the internal quality control are shown in SEQ ID NO.10-11, and the probe sequences are shown in SEQ ID NO.

12.

2. The primer probe combination according to claim 1, characterized in that, The probes for AMB, VZV, HSV-1, and internal quality control are labeled with non-interfering fluorescent groups, and each probe is labeled with a quencher suitable for the corresponding fluorescent group.

3. The primer probe combination of claim 2, wherein, The fluorescent group is selected from any four of FAM, ROX, CY5, VIC, HEX, and TAMRA; the quencher is selected from one or more of BHQ series quenchers and TAMRA.

4. The primer probe combination of claim 3, wherein, The probe corresponding to AMB is labeled with FAM fluorescent group and BHQ1 quencher, the probe corresponding to VZV is labeled with ROX fluorescent group and BHQ2 quencher, the probe corresponding to HSV-1 is labeled with CY5 fluorescent group and BHQ3 quencher, and the probe corresponding to internal quality control is labeled with VIC fluorescent group and BHQ1 quencher.

5. The primer probe combination according to any one of claims 1 to 4, wherein The final concentrations of each primer and probe are as follows: for AMB, VZV, and HSV-1, the final concentrations of the forward and reverse primers are 0.2-0.28 μM; the final concentrations of the corresponding probes are 0.1-0.14 μM; for internal quality control, the final concentrations of the forward primers are 0.14-0.18 μM, the final concentrations of the reverse primers are 0.1-0.14 μM, and the final concentrations of the probes are 0.06-0.10 μM.

6. A detection method for simultaneously detecting AMB, VZV and HSV-1 for non-disease diagnostic purposes, characterized by, Includes the following steps: (1) Nucleic acid extraction from samples; (2) Prepare a PCR reaction system containing any of the primer-probe combinations described in claims 1-5; (3) PCR amplification and result determination.

7. The detection method according to claim 6, characterized in that, The PCR amplification program in step (3) is as follows: incubation at 37℃ for 1-3 min, pre-denaturation at 94-96℃ for 25-35 s; followed by denaturation at 94-96℃ for 4-6 s, annealing and extension at 58-62℃ for 18-22 s, for a total of 38-42 cycles.

8. A reagent kit, characterized in that, It includes the primer-probe combination, PCR premix, internal quality control, positive control and negative control as described in any one of claims 1-5.

9. The use of the primer-probe combination according to any one of claims 1-5 or the kit according to claim 8 in the preparation of products for the diagnosis of ocular infection pathogens, characterized in that, The ocular pathogens are AMB, VZV, and HSV-1.

10. The application according to claim 9, characterized in that, The eye infections include keratitis, retinitis, uveitis, or conjunctivitis.

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