Pathogen detection primer group, kit and application in eye infection pathogen detection

Abstract

The invention discloses a pathogen detection primer group, a kit and application in eye infection pathogen detection, the detection primer group comprises 162 pairs of primers for 54 pathogenic microorganisms, the interference competition among the primers is small, the 54 pathogenic microorganisms can be detected at the same time by randomly selecting one pair of primers for each pathogenic microorganism, and the detection efficiency is high. When all 162 pairs of primers are selected, the sensitivity and specificity of species identification can be improved, the specific and high-sensitivity detection of bacteria, fungi, viruses, parasites and accurately identified species can be met, and the requirements of clinical accurate diagnosis are met.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogen detection, in particular to a pathogen detection primer set, a kit and their application in the detection of eye infection pathogens. Background Art

[0002] Infectious eye diseases are a type of blinding disease caused by pathogenic microorganisms that infect the eyeball and its appendages (conjunctiva, cornea, anterior chamber, vitreous humor, eyelid margin, and lacrimal apparatus), leading to local tissue damage and functional impairment, ultimately causing vision loss. Eye infections are often caused by pathogenic microorganisms such as viruses, bacteria, fungi, and parasites.

[0003] Infectious eye diseases are common clinically, and surgically induced infectious endophthalmitis is also on the rise. The incidence of corneal blindness caused by corneal infection is second only to cataracts. Clinical manifestations and prognosis vary depending on the cause and location of infection. Mild eye infections may only cause ocular discomfort, such as redness, itching, and a foreign body sensation. Severe infections can lead to corneal ulcers, suppuration, perforation, and endophthalmitis, impairing vision and, in severe cases, even leading to blindness and the need for enucleation. Therefore, standardized diagnosis and treatment of infectious eye diseases is crucial. Until now, testing for infectious eye diseases has often lacked a pathological basis, resulting in high rates of missed and misdiagnosis. Treatment based on clinical observation, without precise pathogen detection, often relies on superficial anti-inflammatory treatment rather than targeted, causal relationships. This vicious cycle of misdiagnosis and mistreatment is common, leading to serious consequences for patients' health.

[0004] Currently diagnosed eye diseases include infectious conjunctivitis, infectious keratitis, infectious faciitis, infectious dacryoductitis, and infectious endophthalmitis. Common pathogens causing infectious eye diseases are categorized as viruses, bacteria, fungi, and parasites. The vast majority of common viral infections are caused by human herpes viruses, including the more common herpes simplex virus type 1 (HSV1), varicella zoster virus (VZV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). Less common ones include herpes simplex virus type 2 (HSV2) and human herpes simplex virus (HHV) types 6, 7, and 8. Adenoviruses (types 3 and 7 in children; types 8, 11, and 19 in adults), coxsackievirus A type 24, and enterovirus type 70 can also cause eye infections. Common bacterial infections include Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, and C. acnes. Fungal infections often occur in patients with chronic ocular surface diseases. Common filamentous fungi include Fusarium, Aspergillus, Trichosporon, Paecilomyces, Acremonium, and Curvularia. Parasitic infections are primarily caused by Acanthamoeba, but also include microsporidia, amoebas, and fly larvae.

[0005] Pathogen detection is the gold standard for diagnosing infectious eye diseases. Traditional pathogen detection methods include culture, immunological antigen detection, and PCR. Culture requires specialized laboratories, is costly, time-consuming, and has a low positive rate, hindering timely diagnosis and precise medication. Immunological antigen detection has low specificity and a high false positive / false negative rate. PCR (both conventional PCR and fluorescent quantitative PCR) is limited by the number of pathogens it can detect, limiting comprehensive pathogen testing and easily generating aerosol contamination, leading to a high incidence of false positives.

[0006] Patent document CN112080586A discloses a solid-phase multiplex PCR detection kit and method for infectious eye disease pathogens. The kit uses eight herpes virus primers and probes that have been solid-phase treated and are packaged in triplicate PCR tubes. Each PCR tube contains two to three herpes virus primers and probes with different fluorescent labels, enabling simultaneous detection of eight herpes viruses. However, the detection species are limited to only eight herpes viruses, and the specimens need to be added to three systems. The operation is complicated and a large amount of eye specimens are required.

[0007] Patent document CN118374613A discloses a primer set, a kit, and their applications for detecting intraocular bacterial infection. The qPCR method is used to specifically detect whether a sample contains bacteria. Since the primers are designed within the conserved region sequence of 16S rRNA, the test results can only reflect whether bacteria are present. Clinically, only universal bacterial drug treatment can be performed; targeted treatment cannot be performed if the species cannot be identified.

[0008] Patent document CN115044708A discloses a multiplex real-time fluorescence quantitative PCR kit and method for the simultaneous detection of multiple intraocular infection viruses. Real-time fluorescence quantitative PCR is used to simultaneously detect human herpesvirus type 6 (HHV-6) and rubella virus (RV). This method has high sensitivity and strong specificity, but is limited by the fluorescence channel and can only detect a single species.

[0009] Nanopore sequencing is a next-generation, single-molecule, real-time electrical sequencing technology based on nanopores. It can directly analyze DNA or RNA fragments of any length in real time. It works by monitoring the changes in electrical current as nucleic acids pass through a protein nanopore, decoding these current signals to determine the base sequence. This sequencing technology enables simultaneous sequencing and analysis, significantly reducing sequencing time. However, currently used general barcode identification sequences, such as 16S and ITS, can only accurately identify the genus, but cannot precisely identify the species or even subspecies.

[0010] Based on the current detection methodology for eye infection pathogens, there is an urgent need to develop a detection kit that can simultaneously detect multiple pathogens including bacteria, fungi, viruses, and parasites, and can accurately identify the species. Summary of the Invention

[0011] The present invention provides a pathogen detection primer set to achieve accurate identification of eye infection pathogens capable of simultaneously detecting multiple pathogens including bacteria, fungi, viruses, and parasites, providing a basis for targeted treatment.

[0012] In view of this, the solution of the present invention is: The first aspect of the present invention is to provide a pathogen detection primer set, including primer pairs for 54 pathogens, each pathogen having at least one pair of primers; the specific pathogens and corresponding designed primer sequences are as follows:

[0013] Preferably, the primer set includes all 162 pairs of primers for 54 different pathogens whose nucleotide sequences are shown as SEQ ID NOs: 1 to 324.

[0014] The second aspect of the present invention is to provide the use of the primer set described in the first aspect in preparing a product for detecting ocular infection pathogens.

[0015] The third aspect of the present invention is to provide an eye infection pathogen detection kit, comprising the primer set described in the first aspect.

[0016] Furthermore, in the above kit, a common sequence is added to the 5' end of each primer sequence in the primer set, and the kit also includes a barcode sequence tag for distinguishing different amplicons.

[0017] Furthermore, the kit further comprises at least one of a nucleic acid extraction reagent, a multiplex PCR reaction reagent, a barcode ligation PCR reaction reagent, and a nanopore library construction reagent.

[0018] Preferably, the final concentration of each primer pair in the primer set is 5-100 nM.

[0019] The fourth aspect of the present invention is to provide use of the primer set described in the first aspect or the kit described in the third aspect in pathogen detection, wherein the use is for non-diagnostic purposes.

[0020] The fifth aspect of the present invention is to provide a method for preparing pathogen sequencing fragments, comprising the step of performing PCR amplification on the sample nucleic acid using the primer set described in the first aspect.

[0021] The sixth aspect of the present invention is to provide a method for detecting pathogens for non-diagnostic purposes, the steps including amplifying the sample nucleic acid with the primer set described in the first aspect, then sequencing the amplicon, and comparing the sequencing result with the reference gene to obtain the detection result.

[0022] Furthermore, the amplification process includes performing a first round of PCR amplification on the sample nucleic acid using a primer set, and connecting the barcode to perform a second round of PCR amplification; the first round of PCR amplification adds a common sequence to the 5' end of each primer in the primer set; And / or, the sequencing is based on nanopore sequencing.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The detection primer set provided by the present invention contains 162 pairs of primers for 54 pathogenic microorganisms. The interference competition between the primers is small. By selecting any one pair of primers for each pathogenic microorganism, 54 pathogenic microorganisms can be detected simultaneously. When all 162 pairs of primers are selected, the sensitivity and specificity of species identification can be improved, and the specificity and high sensitivity detection of bacteria, fungi, viruses, and parasites can be accurately identified to the species level, meeting the needs of clinical precision diagnosis.

[0024] The detection kit provided by this invention can accurately identify 54 common pathogenic microorganisms in ophthalmic infections. The long read length of nanopore sequencing can be combined with this kit for even more accurate identification. Compared to conventional PCR and qPCR, it can detect more targets, has higher throughput, and lowers testing costs. The detection process is simple and quick, with results available within 6 hours. The sequencing instrument is compact and portable, enabling sequencing and testing anytime, anywhere. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0026] The technical solutions of the present invention will be clearly and completely described below in conjunction with preferred embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] In one embodiment, a super-multiplex PCR system combined with nanopore targeted sequencing technology is proposed. Specific primer sets are designed for pathogenic microorganisms such as bacteria, fungi, viruses, and parasites that can cause eye infections. A super-multiplex PCR detection system is constructed to amplify 54 common eye infection pathogens in a single reaction tube. The amplified pathogens are sequenced using a nanopore sequencing platform. Through bioinformatics comparison, pathogen detection results are quickly and accurately obtained, assisting clinical diagnosis and guiding precision medication. This is a diagnostic method with application prospects. In addition, the reagents provided in this kit can be used with different nanopore sequencing platforms, not limited to Oxford Nanopore's ONT sequencing platform, Puyi Bio's Polyseq sequencing platform, MGI's Cycloneseq and other domestic sequencing platforms.

[0028] In the above example, this technology can simultaneously and accurately identify 54 common pathogens of ocular infections, with results available within 6 hours. It offers high throughput, low cost, and simple operation. The compact and portable sequencing instrument allows for sequencing and testing anytime, anywhere. The 54 pathogens are listed in Table 1.

[0029] Table 1:

[0030] In the above embodiment, the specific primer set includes primer pairs for 54 pathogens, and each pathogen is provided with at least one pair of primers; the specific pathogens and the corresponding designed primer sequences are shown in Table 2.

[0031] Table 2:

[0032] The primer sets shown in Table 2 above contain three available primer pairs for each species. While any one of these three pairs has been shown to be sufficient for detection of the pathogenic species, using them simultaneously can improve the sensitivity and specificity of species identification. Simultaneous use of all three pairs for each species, totaling 162 primer pairs, has been shown to exhibit no cross-reactivity, minimal competition from primer sequence interference, and excellent overall detection sensitivity and specificity.

[0033] Example 1 Selection of target species for ophthalmic infection pathogens

[0034] The target species for ophthalmic infections were selected mainly by collecting and collating a large number of domestic and foreign literature, websites and target species of ophthalmic infection-related pathogens on the market. Finally, a set of common pathogens that can cause ophthalmic infections was determined, including the 54 pathogens shown in Table 1.

[0035] Example 2 Design and verification of 54 pathogen-specific primers

[0036] Primer pool design: All high-quality genome sequences of 54 pathogens and humans were downloaded from the NCBI database. Through bioinformatics comparison analysis, specific sequences that were conserved within and specific between species were selected for each pathogen. Primers were designed using Primer 5.0, with primers with the highest scores being prioritized. Five to ten primer pairs were designed for each pathogen, ranked according to the primer design score. The top three primer pairs from the first species served as the initial primer pool. Primers from additional species were subsequently added to the pool. Primer dimer analysis was performed on these primers compared to existing primers in the pool. Primers that were found to be prone to primer dimer formation were discarded, and alternative primers were selected. The final primer pool should contain one to three specific primer pairs for each pathogen.

[0037] Experimental verification: 54 kinds of pathogenic microorganisms were established as reference materials for the company. All primers in the primer pool were amplified and sequenced. Primers with no amplification bands or cross-reactions were eliminated. Primers were redesigned and supplemented and experimentally verified. After a large number of repeated experimental tests, the primer pool shown in Table 3 was finally obtained. Figure 1 This is a gel image of the corresponding primers. Each species in the primer pool has 1-3 pairs of available primers. It has been demonstrated that any one of these 1-3 pairs can detect the pathogen, but using them together can improve the sensitivity and specificity of species identification. The primer pool, comprising 162 pairs, has been experimentally demonstrated to exhibit no cross-reactivity, minimal primer sequence interference, and excellent overall detection sensitivity and specificity.

[0038] Table 3:

[0039]

[0040]

[0041]

[0042] Example 3 Primer combination for detection of ophthalmic infection pathogens

[0043] A single round of reactions uses a multiplexed specific reaction sequence combination. Each sequence consists of a segment a and a segment b from the 5' to 3' end. The segment a is a common sequence, and the segment b is the forward and reverse sequences of 162 pairs of specific primers (SEQ ID NOs: 1-324). A total of 324 sequences are combined and used at specific concentrations. The final concentration of each primer system ranges from 5 nM to 100 nM, with 20 nM being preferred. Table 4 shows three primer combinations for Toxoplasma gondii as examples.

[0044] Table 4:

[0045] The underlined sequence in Table 4 is the common sequence of segment a.

[0046] Example 4 Kit Composition

[0047] Nanopore sequencing has a high cost for single library construction and sequencing, but it can detect multiple samples at the same time in one sequencing, which can share the cost and significantly reduce the detection of single samples. This kit is designed with 96 barcode sequence tags, which are connected to a round of amplification-specific products through a common sequence. A different barcode can be selected for each sample. 96 samples can be detected simultaneously in one sequencing, but it is not limited to detecting 96 samples. If there is a need for detection, it can be expanded to 384 samples. This embodiment provides a kit for the detection of ophthalmic infection pathogens based on targeted nanopores, including multiple PCR reaction reagents, barcode connection PCR reaction reagents and ONT library construction reagents. The components of the kit are shown in Table 5.

[0048] Table 5:

[0049] It is understandable that in high-throughput sequencing, barcodes are mainly used to distinguish different samples, usually by adding them to the ends of primers. The addition of the barcode sequence itself does not directly affect the amplification of the primers.

[0050] Example 5 Method for detecting common pathogens of ophthalmic infections

[0051] (1) Sample pretreatment and nucleic acid extraction

[0052] It is recommended to use nucleic acid extraction or purification reagents (Beina Medical, TQ009D-64, TQ010D-64). Please refer to the corresponding instructions for specific extraction methods.

[0053] (2) One round of multiple target-specific reactions

[0054] The extracted nucleic acid and positive and negative quality controls were subjected to multiplex PCR amplification according to the reaction system in the following table: The one-round multiplex amplification system of this kit is as follows:

[0055] Reaction conditions: After experimental testing, the annealing temperature is 53~60℃, preferably 60℃; the annealing time is 30s~2min, preferably 1min; the extension time is 30s~2min, preferably 1min:

[0056] (3) Second round of barcode ligation PCR reaction

[0057] Take one round of reaction products and perform barcode ligation PCR reaction according to the table below. Different barcode sequence primers are selected for different samples:

[0058] Reaction conditions: After experimental testing, the annealing temperature is 55~65℃, preferably 60℃; the annealing time is 30s~2min, preferably 30s; the extension time is 30s~1min, preferably 40s:

[0059] (4) Mixing and purification

[0060] 4.1 Take a new 1.5 ml EP tube and mix equal volumes of the barcode-linked PCR products.

[0061] 4.2 Resuspend AMPure XP magnetic beads by vortexing.

[0062] 4.3 Take 200 μl of the mixed PCR product, add 140 μl of AMPure XP magnetic beads, mix by flicking the tube, and incubate at room temperature for 5 minutes.

[0063] 4.4 Place the EP tube on the magnetic stand until the eluate becomes clear and colorless, then remove the supernatant.

[0064] 4.5 Place the EP tube on the magnetic stand and wash the magnetic beads with 200 μl of freshly prepared 80% ethanol. Remove and discard the ethanol.

[0065] 4.6 Repeat the previous step.

[0066] 4.7 Centrifuge briefly and place the EP tube back on the magnet to remove any remaining ethanol. Allow to dry for approximately 30 seconds, but do not dry to the point of cracking the pellet.

[0067] 4.8 Remove the EP tube from the magnetic stand, resuspend the magnetic beads in 52 μl of EP, and incubate at room temperature for 2 minutes.

[0068] 4.9 Place the EP tube on the magnetic rack until the eluent becomes transparent and colorless.

[0069] 4.10 Transfer all supernatant to a new 1.5 ml EB tube.

[0070] 4.11 Take 1 μl of the purified product and measure the concentration using the Qubit dsDNA HS Assay Kit.

[0071] (5) Connector ligation and purification

[0072] 5.1 Configure the adapter ligation system. Different nanopore sequencing platforms can use the optimized ligation system in this kit for adapter ligation. You only need to replace the adapter mix of different nanopore sequencing platforms during the ligation process.

[0073]

[0074] 5.2 Place the PCR reaction tubes on the PCR instrument; program: 24°C for 10 minutes. (Reaction time: 10-30 minutes. To shorten the assay, 10 minutes is sufficient; to improve the reaction quality, the reaction time can be extended appropriately.)

[0075] 5.3 Transfer the PCR product to a new 1.5 ml EP tube, add 80 μl AMPure XP magnetic beads, and perform magnetic bead purification according to the method in step (3). Replace 80% ethanol with pH for washing, and elute with 20 μl EB to obtain purified DNA.

[0076] 5.4 Take 1 μl of the purified product and measure the concentration using the Qubit dsDNA HS Assay Kit.

[0077] (6) Sequencing

[0078] This test kit can be used with various nanopore sequencing platforms, including but not limited to Oxford Nanopore's ONT sequencing platform, as well as domestic nanopore sequencing platforms such as Puyi Biotech, MGI, and Jinshi Technology. Follow the instructions for the nanopore sequencer to operate the instrument.

[0079] (7) Bioinformatics Analysis

[0080] Perform bioinformatics analysis on the offline data, compare it with the pathogen database, and analyze the test results.

[0081] Example 6

[0082] Enterprise reference materials for 54 strains were established, and the Lod detection limit and cross-reaction test of some negative samples were tested using the above method. The test results are shown in Table 6.

[0083] Table 6:

[0084]

[0085]

[0086]

[0087]

[0088] The test results showed that all primers for 54 species could be effectively amplified, with the detection Lod ranging from 50 to 1000 copies / mL. There was no cross-reaction with other species of the same genus that were not within the detection range, and the detection was negative.

[0089] Example 7

[0090] Positive samples tested using the gold standard method for clinical pathogen detection (culture or qPCR) were tested using the above method, and the test results are as follows:

[0091]

[0092] Comparing the results of different detection methods, the results of the present invention were 100% consistent with those of the culture method or qPCR, indicating that the present method can effectively detect pathogens of eye infections.

[0093] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A pathogen detection primer set, characterized in that: Includes primer pairs for 54 pathogens, with at least one pair of primers for each pathogen; the specific pathogens and corresponding designed primer sequences are as follows: 。 2. Use of the primer set according to claim 1 in preparing a product for detecting ocular infection pathogens.

3. A kit for detecting eye infection pathogens, characterized in that: Comprising the primer set according to claim 1.

4. The kit according to claim 3, wherein A common sequence is added to the 5' end of each primer sequence in the primer set, and the kit further comprises a barcode sequence tag for distinguishing different amplicons.

5. The kit according to claim 3, characterized in that The kit further comprises at least one of a nucleic acid extraction reagent, a multiplex PCR reaction reagent, a barcode ligation PCR reaction reagent, and a nanopore library construction reagent.

6. The kit according to claim 4, wherein The final concentration of each primer pair in the primer set is 5-100 nM.

7. Use of the primer set according to claim 1 or the kit according to any one of claims 3 to 6 in pathogen detection, wherein the use is for non-diagnostic purposes.

8. A method for preparing pathogen sequencing fragments, characterized in that: The method comprises the step of performing PCR amplification on the sample nucleic acid using the primer set according to claim 1.

9. A method for detecting pathogens for non-diagnostic purposes, characterized in that: The steps include amplifying the sample nucleic acid with the primer set of claim 1, then sequencing the amplicon, and comparing the sequencing result with the reference gene to obtain the test result.

10. The method according to claim 8, characterized in that The amplification process includes performing a first round of PCR amplification on the sample nucleic acid using a primer set, and connecting a barcode to perform a second round of PCR amplification; the first round of PCR amplification adds a common sequence to the 5' end of each primer in the primer set; And / or, the sequencing is based on nanopore sequencing.

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