Pathogen detection primer set, kit and application in detection of pathogen in eye infection

Abstract

The application discloses a pathogen detection primer group, a kit and application in pathogen detection of eye infection, the detection primer group contains 162 pairs of primers for 54 pathogenic microorganisms, interference competition between each primer is small, and one pair of primers is selected for each pathogenic microorganism, so that the detection of the 54 pathogenic microorganisms can be realized simultaneously, when all the 162 pairs of primers are selected, the sensitivity and specificity of species identification can be improved, specific identification to species and high-sensitivity detection of bacteria, fungi, viruses and parasites can be realized, and the requirement of precise clinical diagnosis can be met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pathogen detection, in particular to a pathogen detection primer set, a kit and application in detection of ocular infection pathogens. BACKGROUND

[0002] Infectious eye disease is a kind of blinding disease caused by pathogenic microorganisms infecting ocular tissues and their appendages (conjunctiva, cornea, anterior chamber, vitreous body, eyelid margin and lacrimal apparatus, etc.), leading to local tissue damage, functional damage, and further causing visual impairment. Ocular infection is mostly caused by pathogenic microorganisms such as viruses, bacteria, fungi and parasites.

[0003] Infectious eye disease is a common eye disease in clinic, and surgical infectious endophthalmitis also has an upward trend. The incidence of corneal blindness caused by corneal infection is only second to cataract. Due to the difference in infection causes and sites, the clinical manifestations and prognosis are also different. Mild eye infection only causes eye discomfort symptoms such as eye redness, eye itching and foreign body sensation, while severe infection can cause corneal ulcer, suppuration, perforation and endophthalmitis, etc., thereby affecting the visual acuity of patients, and even causing blindness and enucleation of the eyeball in severe cases. Therefore, it is particularly important to standardize the diagnosis and treatment of ocular infectious diseases. So far, the detection of infectious eye disease often lacks pathogenic basis in the diagnosis and treatment process, and the misdiagnosis rate and misdiagnosis rate are undoubtedly high. Based on clinical observation, the treatment lacks accurate pathogen detection, and more is the anti-inflammatory treatment based on the appearance, rather than the targeted treatment based on the cause and effect. Misdiagnosis and mistreatment are common, and such diagnosis and treatment practice has serious consequences for the health of patients.

[0004] Current ocular diagnostic diseases include infectious conjunctivitis, infectious keratitis, infectious blepharitis, infectious lacrimal duct inflammation, infectious endophthalmitis, etc. The common pathogens causing infectious eye disease are divided into viruses, bacteria, fungi and parasites. The most common viral pathogens are human herpes viruses, among which the more common ones are herpes simplex virus type 1 (HSV1), varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and the less common ones are herpes simplex virus type 2 (HSV2) and human herpes virus (HHV) 6, 7, 8, etc. In addition, adenovirus (children 3, 7 type; adults 8, 11, 19 type), coxsackie virus A 24 type, enterovirus 70 type can also cause ocular infection. Common bacterial pathogens include Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Haemophilus influenzae, and Acne skin bacillus, etc. Fungal infection often occurs in patients with chronic ocular surface diseases. Common filamentous fungi include fusarium, aspergillus, hyalohyphomycetes, paecilomyces, camposporium and curvularia, etc. Parasitic infection is mainly caused by acanthamoeba infection, in addition to microsporidium, amoeba and fly larva.

[0005] The pathogenic microorganism examination is the gold standard for the diagnosis of infectious eye diseases. Traditional pathogen detection methods include culture method, immunological detection of antigen method, PCR technology, etc. Among them, the culture method needs a special culture laboratory, has high cost, time-consuming process and low positive rate, which is not conducive to timely diagnosis and precise medication. The immunological detection of antigen method has low specificity and high false positive / false negative. The PCR technology (ordinary PCR and fluorescent quantitative PCR) is limited by the type of detected pathogen, the detection species is single, and it cannot be used for comprehensive pathogen examination, and it is easy to produce aerosol pollution, resulting in many false positive results.

[0006] Patent document CN112080586A discloses an infectious eye disease pathogen solid-phase multiple array PCR detection kit and detection method, which uses 8 kinds of herpes virus primers and probes after solid-phase treatment, and is divided into three array PCR tubes. Each PCR tube contains 2-3 kinds of herpes virus primers and probes with different fluorescent labels, which can simultaneously detect 8 kinds of herpes viruses, but the detection species is limited to only eight kinds of herpes viruses, and the specimen needs to be added into three systems, which is complex to operate and requires a large amount of eye specimen.

[0007] Patent document CN118374613A discloses a primer set, kit and application for detecting intraocular bacterial infection, which uses qPCR method to specifically detect whether the sample contains bacteria. Since the primers are designed based on the conserved region sequence of 16S rRNA, the detection result can only reflect whether the sample contains bacteria, which can only be used for general bacterial drug treatment in clinical practice, and cannot be used for targeted treatment.

[0008] Patent document CN115044708A discloses a multiple real-time fluorescent quantitative PCR kit and method for simultaneously detecting multiple intraocular infection viruses, which uses real-time fluorescent quantitative PCR to simultaneously detect human herpes virus 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 new generation of nanopore-based single-molecule real-time electrical signal sequencing technology. It can directly and real-time analyze DNA or RNA fragments of any length. It works by real-time monitoring of the current change when nucleic acid passes through the protein nanopore, and decodes these current signals to determine the base sequence. This sequencing technology can analyze while sequencing, greatly shortening the sequencing time. However, most of the commonly used 16S, ITS, etc. universal barcode identification sequences can accurately identify to the genus level, but cannot accurately identify to the species level, or even subspecies level.

[0010] Based on the current detection methods of eye infection pathogens, it is urgent to develop a detection kit that can simultaneously detect multiple pathogens including bacteria, fungi, viruses, and parasites, and accurately identify to the species level. SUMMARY

[0011] The present application provides a pathogen detection primer set, which can be used to simultaneously detect multiple pathogens including bacteria, fungi, viruses and parasites, and to accurately identify the pathogens of eye infections, thereby providing a basis for targeted treatment.

[0012] Therefore, the present application provides the following solutions:

[0013] In a first aspect, the present application provides a pathogen detection primer set, which comprises primer pairs for 54 pathogens, at least one primer pair for each pathogen, and the specific pathogens and the corresponding designed primer sequences are as follows:

[0014]

[0015] Preferably, the primer set comprises all 162 primer pairs for 54 different pathogens, and the nucleotide sequences of the primer pairs are as shown in SEQ ID NO: 1-324.

[0016] In a second aspect, the present application provides the use of the primer set of the first aspect in the preparation of a pathogen detection product for eye infections.

[0017] In a third aspect, the present application provides a pathogen detection kit for eye infections, which comprises the primer set of the first aspect.

[0018] Further, in the above-mentioned kit, 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 label for distinguishing different amplicons.

[0019] Further, 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.

[0020] Preferably, the final concentration of each primer pair in the primer set is 5-100 nM.

[0021] In a fourth aspect, the present application provides the use of the primer set of the first aspect or the kit of the third aspect in pathogen detection, which is not for diagnostic purposes.

[0022] In a fifth aspect, the present application provides a method for preparing a pathogen sequencing fragment, which comprises the step of performing PCR amplification on a sample nucleic acid using the primer set of the first aspect.

[0023] The sixth aspect of the present application provides a method for detecting pathogens, for non-diagnostic purposes, comprising amplifying the nucleic acid of the sample with the primer set of the first aspect, then sequencing the amplicon, comparing the sequencing results with the reference gene, and obtaining the detection results.

[0024] Further, the amplification process comprises first round PCR amplification of the sample nucleic acid using the primer set, and second round PCR amplification of the barcode; the first round PCR amplification adds a common sequence to the 5' end of each primer in the primer set;

[0025] And / or, the sequencing is based on nanopore sequencing method.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The detection primer set provided by the present application contains 162 pairs of primers for 54 pathogenic microorganisms, the interference competition between each primer is small, and one pair of primers can be selected for each pathogenic microorganism to realize the detection of 54 pathogenic microorganisms at the same time, when all the 162 pairs of primers are selected, the sensitivity and specificity of species identification can be improved, the specificity and high sensitivity detection of accurate identification to species including bacteria, fungi, viruses and parasites can be met, and the demand of clinical precise diagnosis can be met.

[0028] The detection kit provided by the present application can accurately identify 54 common pathogenic microorganisms in ophthalmic infections, can be combined with the long read characteristics of nanopore sequencing when used for detection, and the identification is more accurate; compared with ordinary PCR and qPCR, the detection targets are more, the throughput is higher, and the detection cost is lower. The detection process is simple and short in time, and the results can be obtained within 6 hours. The sequencing instrument is small and portable, and can realize sequencing detection anytime and anywhere. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The schematic diagram is described in the present application. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described below in conjunction with preferred embodiments, obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0031] In one embodiment, a super-multiplex PCR system combined with nanopore target 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, and 54 common eye infection pathogens are amplified in one reaction tube. Sequencing is performed through a nanopore sequencing platform. Through bioinformatics comparison, the pathogen detection results are quickly and accurately obtained, which assists clinical diagnosis and guides precise drug use. It is a diagnostic method with application prospect. In addition, the reagents provided in the kit can be combined with different platform nanopore sequencing platforms, not limited to the ONT sequencing platform of Oxford Nanopore, the Polyseq sequencing platform of Polygenetech, and the Cycloneseq domestic sequencing platform of Huada.

[0032] In the above embodiment, the technology can simultaneously accurately identify 54 common pathogenic microorganisms in eye infections, and the results can be obtained within 6 hours. The technology has high throughput, low cost, and simple operation. The sequencing instrument is small and portable, and can be used for sequencing detection anytime and anywhere. The 54 pathogenic microorganisms are shown in Table 1.

[0033] Table 1:

[0034]

[0035] In the above embodiment, the specific primer set includes primer pairs for 54 pathogens, and at least one pair of primers is provided for each pathogen. The specific primers and corresponding primer sequences for each pathogen are shown in Table 2.

[0036] Table 2:

[0037]

[0038] In the above embodiment, the specific primer set includes primer pairs for 54 pathogens, and at least one pair of primers is provided for each pathogen. The specific primers and corresponding primer sequences for each pathogen are shown in Table 2.

[0039] Example 1: Selection of eye infection pathogen target species

[0040] The selection of eye infection target species is mainly through collecting and sorting a large number of domestic and foreign literature, websites, and market-related eye infection pathogen target species, and finally determining the common pathogen collection that can cause eye infection, including 54 pathogens shown in Table 1.

[0041] Example 2: Design and verification of 54 pathogen-specific primers

[0042] Primer pool design: Download all good quality genome sequences of 54 pathogenic microorganisms and humans from the NCBI database, analyze by bioinformatics comparison, select specific sequences within each species and specific to other species, use primer 5.0 to design primers, and preferentially select primers with high scores. Design 5-10 pairs of primers for each pathogen, sort according to primer design score, and use the first three pairs of primers of the first species as the initial primer pool, and continuously add primers from other species. Primer dimer analysis is performed on the primers in the primer pool, and if the primers that are prone to generate primer dimers are removed, other standby primers are selected, and the final primer pool needs to meet the requirement of 1-3 pairs of specific primers for each pathogen.

[0043] Experimental verification: Establish enterprise reference materials for 54 pathogenic microorganisms, amplify and sequence all primers in the primer pool, and remove primers with no amplification bands or cross-reactions. Redesign primers for supplementation and experimental verification. Through a large number of repeated experiments, the primer pool in Table 3 is finally obtained, Figure 1 is the gel map of the corresponding primers. Each species in the primer pool has 1-3 pairs of available primers, and it has been proven that using any one of the 1-3 pairs of primers can achieve the detection of the pathogen species, but simultaneous use can improve the sensitivity and specificity of species identification. A total of 162 pairs of primers make up the primer pool, which has been proven by experiments to have no cross-reactions with each other, the primer sequence has little interference and competition, and the overall detection sensitivity and specificity are very good.

[0044] Table 3:

[0045]

[0046]

[0047]

[0048]

[0049] Example 3 Ophthalmic infection pathogen detection primer combination

[0050] The multiplex specific reaction sequence combination used in one round of reaction is composed of a segment sequence and a segment sequence from 5' end to 3' end, wherein the a segment sequence is a common sequence, and the b segment sequence is the forward and reverse sequences of 162 pairs of specific primers (SEQ ID NO: 1~324). A total of 324 sequences are obtained by combination, and are mixed and used according to a certain concentration. The final concentration of each primer system is 5nM~100nM, preferably 20nM concentration, and Table 4 gives 3 pairs of primers of Naegleria gruberi as an example.

[0051] Table 4:

[0052]

[0053] The underlined sequences in Table 4 are the common sequences of section a.

[0054] Kit composition of Example 4

[0055] The single library sequencing cost of nanopore sequencing is high, and multiple samples can be detected at the same time, the cost is shared, and the single sample detection is significantly reduced. The kit designed 96 barcode sequence tags, which are connected to the specific product of one round of amplification through common sequences, and one different barcode can be selected for each sample, 96 samples can be detected at the same time, but it is not limited to detecting 96 samples. If there is a detection demand, it can be expanded to 384 samples. The present embodiment provides a kit for detecting ophthalmic infection pathogens based on targeted nanopore, which comprises multiplex PCR reaction reagent, barcode connection PCR reaction reagent and ONT library construction reagent. The kit components are shown in Table 5.

[0056] Table 5:

[0057]

[0058] It can be understood that in high-throughput sequencing, barcode is mainly used to distinguish different samples, and the addition of barcode sequence itself will not directly affect the amplification of primer, usually by adding to the end of primer.

[0059] Method for detecting common pathogens of ophthalmic infections of Example 5

[0060] (1) Sample pretreatment and nucleic acid extraction

[0061] It is recommended to use nucleic acid extraction or purification reagent (Benay Medical, TQ009D-64, TQ010D-64). For specific extraction method, please refer to the corresponding instruction manual.

[0062] (2) One round of multiplex targeted specific reaction

[0063] The extracted nucleic acid and positive and negative controls are subjected to multiplex PCR amplification according to the following reaction system:

[0064] The one round of multiplex amplification system of the kit is as follows:

[0065]

[0066] The reaction conditions are as follows: the annealing temperature is 53-60℃, preferably 60℃; the annealing time is 30s-2min, preferably 1min; and the extension time is 30s-2min, preferably 1min:

[0067]

[0068] (3) Two rounds of barcode ligation PCR reaction

[0069] Take the first round of reaction product to carry out barcode ligation PCR reaction according to the following table, and select different barcode sequence primers for different samples:

[0070]

[0071] The reaction conditions are tested by experiments, and the annealing temperature is 55-65°C, preferably 60°C; the annealing time is 30s-2min, preferably 30s; the extension time is 30s-1min, preferably 40s:

[0072]

[0073] (4) Mixing and purification

[0074] 4.1 Take a new 1.5 ml EP tube, and mix the PCR product with ligation barcode in equal volume.

[0075] 4.2 Resuspend the AMPure XP magnetic beads by vortexing.

[0076] 4.3 Take 200 μl of mixed PCR product, add 140 μl of AMPure XP magnetic beads, mix by tapping the EP tube, and incubate at room temperature for 5 minutes.

[0077] 4.4 Place the EP tube on the magnetic stand until the eluate is clear and colorless, then remove the supernatant.

[0078] 4.5 Place the EP tube on the magnetic stand, wash the magnetic beads with 200 μl of freshly prepared 80% ethanol, remove the ethanol and discard.

[0079] 4.6 Repeat the previous step.

[0080] 4.7 Centrifuge momentarily and place the EP tube back on the magnetic stand, remove all residual ethanol. Dry for about 30 seconds, but do not dry to the point of cracking the particles.

[0081] 4.8 Take the EP tube from the magnetic stand, resuspend the magnetic beads in 52 μl of EP, and incubate at room temperature for 2 minutes.

[0082] 4.9 Place the EP tube on the magnetic stand until the eluate is clear and colorless.

[0083] 4.10 Take all the supernatant to a new 1.5 ml EB tube.

[0084] 4.11 Take 1 μl of purified product to detect the concentration with Qubit dsDNA HS Assay Kit.

[0085] (5) Connector connection and purification

[0086] 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 for different nanopore sequencing platforms during the ligation process.

[0087]

[0088] 5.2 Place the PCR reaction tubes on the PCR instrument; program: 24℃ for 10 min. (Reaction time is 10 min-30 min. To shorten the detection time, 10 min is sufficient. If the reaction effect is to be improved, the reaction time can be appropriately extended.)

[0089] 5.3 Transfer the PCR product to a new 1.5ml EP tube, add 80 μl of 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 of EB to obtain purified DNA.

[0090] 5.4 Take 1 μl of the purified product and use the Qubit dsDNA HS Assay Kit to detect its concentration.

[0091] (6) Sequencing

[0092] This test kit can be used with various nanopore platforms for sequencing, including but not limited to the Oxford Nanopore ONT sequencing platform, as well as all domestic nanopore sequencing platforms such as Puyi Bio, BGI Genomics, and Jinshi Technology. Follow the nanopore sequencer's instruction manual for operation.

[0093] (7) Bioinformatics analysis

[0094] Bioinformatics analysis was performed on the data from the test, and the results were compared with the pathogen database.

[0095] Example 6

[0096] Fifty-four strains were established as enterprise reference samples. The detection limit of LOD and cross-reactivity of some negative samples were tested using the above method. The test results are shown in Table 6.

[0097] Table 6:

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] The detection results showed that all primers of 54 species could be effectively amplified, the detection Lod was 50-1000 copies / mL, and there was no cross reaction with other same genus species out of the detection range, and the detection was negative.

[0104] Example 7

[0105] The positive samples detected by the clinical pathogen detection gold standard method (culture or qPCR) were detected by the above method, and the detection results were as follows:

[0106]

[0107]

[0108] Comparing the results of different detection methods, the consistency of the present application and the culture method or qPCR was 100%, indicating that the present method could effectively detect the ocular infection pathogens.

[0109] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A pathogen detection primer set, characterized by, The primer set comprises primer pairs for 54 pathogens, at least one pair of primers for each pathogen; the specific pathogens and the corresponding designed primer sequences are as follows: 。 2. The primer set of claim 1 is applied to the preparation of an ocular infection pathogen detection product.

3. An eye infection pathogen detection kit characterized by, The primer set comprises the primer set of claim 1.

4. The kit of 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 label for distinguishing different amplicons.

5. The kit of claim 3, wherein 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 of claim 4, wherein The final concentration of each primer pair in the primer set is 5-100 nM.

7. The primer set of claim 1 or the kit of any one of claims 3-6 is applied to pathogen detection, and the application is for non-diagnostic purposes.

8. A method for preparing a pathogen sequencing fragment, characterized in that, The primer set comprises the steps of using the primer set of claim 1 to perform PCR amplification on the nucleic acid of the sample.

9. A method of detecting a pathogen for non-diagnostic purposes, characterized in that, The steps comprise amplifying the nucleic acid of the sample using the primer set of claim 1, then sequencing the amplicon, and comparing the sequencing results with reference genes to obtain the detection results.

10. The method of claim 9, wherein, The amplification process comprises first round PCR amplification of the sample nucleic acid using the primer set, and second round PCR amplification of the barcode ligation; the first round PCR amplification adds a common sequence to the 5' end of each primer in the primer set; And / or, the sequencing is based on the nanopore sequencing method.

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