Multiple bacterial and fungal nucleic acid detection primer probe set, kit, microfluidic chip and application

Abstract

The application provides a multiple bacterial and fungal nucleic acid detection primer probe set, a kit, a microfluidic chip and application; 47 pairs of primers and Toehold strand displacement fluorescent probes are designed for 47 kinds of bacterial and fungal pathogens; specific amplification is completed in a single tube by the primer pairs; the Toehold strand displacement fluorescent probes can efficiently generate a chain displacement reaction with the target to produce a fluorescent signal; the primer pairs and the Toehold strand displacement fluorescent probes can avoid the interference problem of multiple amplification, and can efficiently detect any bacterial and fungal pathogens in a single tube, and have significant value for environmental sample or clinical sample detection.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection kit technology, specifically to multiplex bacterial and fungal nucleic acid detection primer and probe sets, kits, microfluidic chips, and applications. Background Technology

[0002] Mixed bacterial and fungal infections are increasingly common in clinical practice, particularly in immunocompromised patients, those in intensive care, and those on long-term antibiotic treatment. Rapid and accurate identification of pathogens is crucial for guiding anti-infective therapy and reducing mortality. However, current detection technologies have significant limitations in terms of detection efficiency and multiplexing capabilities.

[0003] While traditional culture methods are considered the "gold standard," they are time-consuming, taking several days, and have low detection rates for fastidious bacteria or fungi. Conventional PCR and real-time quantitative PCR reduce detection time to 1-2 hours, but still rely on sophisticated thermal cycling equipment, making truly rapid point-of-care or field testing difficult. Multiplex PCR, although capable of detecting multiple targets simultaneously, suffers from significant primer dimerization and non-specific amplification issues, especially when targets contain genomes of bacteria and fungi that differ considerably. The reaction system is prone to imbalance and typically requires multi-channel fluorescence equipment or subsequent electrophoresis analysis, making the process cumbersome.

[0004] Convection PCR, a rapid thermal cycling technique based on natural convection, can significantly shorten detection time. However, its application in multiplex detection is still limited by the single signal output mode and spatial resolution of conventional fluorescent probes. Existing methods mostly use the number of fluorescent channels corresponding to the number of targets, making it difficult to overcome the bottleneck of simultaneously detecting multiple bacteria and fungi in a single tube. In addition, although probe hybridization substitution technology has shown high specificity in the detection of single nucleotide polymorphisms, it has not yet been effectively integrated with convection PCR for multiplex pathogen detection.

[0005] Currently available reagent kits rarely achieve simultaneous detection of bacteria and fungi in a single tube, and most require reaction times of over one hour and complex equipment. Therefore, developing a reagent kit that can complete the combined detection of multiplex bacteria and fungi in a single tube in a short time would overcome the speed and instrument limitations of traditional PCR and solve the interference problem of multiplex amplification, showing significant application potential. Summary of the Invention

[0006] This invention proposes a primer and probe set for the detection of multiplex bacteria and fungi nucleic acids, which can be used to specifically amplify and detect multiple bacteria and fungi simultaneously in a single tube. Combined with convective PCR technology, it can complete the specific detection of multiple bacteria and fungi in a short time.

[0007] The technical solution of this invention is implemented as follows: The first aspect of the present invention is to provide a primer and probe set for the detection of multiplex bacterial and fungal nucleic acids, comprising a primer set with nucleotide sequences as shown in SEQ ID NO. 1-94, and a Toehold chain substitution fluorescent probe set; the Toehold chain substitution fluorescent probe set includes an Arm probe, a Quencher probe, and an Anchor probe; the Arm probe and the Quencher probe correspond to the odd and even sequences in the nucleotide sequences shown in SEQ ID NO. 97-190, respectively, and the nucleotide sequence of the Anchor probe is shown in SEQ ID NO. 193.

[0008] Furthermore, the nucleic acid detection primer and probe set also includes an internal reference gene detection primer pair and a fluorescent probe set; preferably, the internal reference gene is the GAPDH gene.

[0009] Furthermore, the primer set also includes internal reference gene detection primer pairs with nucleotide sequences as shown in SEQ ID NO. 95-96, and the Toehold chain substitution fluorescent probe set also includes internal reference Arm probe and internal reference Quencher probe with nucleotide sequences as shown in SEQ ID NO. 191 and 192, respectively.

[0010] A second aspect of the present invention is to provide the application of the nucleic acid detection primer and probe set described in the first aspect in the preparation of multiplex bacterial and fungal nucleic acid detection products.

[0011] Furthermore, the testing products include, but are not limited to, reagent kits, chips, and testing instruments.

[0012] A third aspect of the present invention is a multiplex bacterial and fungal nucleic acid detection kit, which includes the nucleic acid detection primer and probe set described in the first aspect.

[0013] Furthermore, the test kit also includes at least one of the following: nucleic acid extraction reagent, amplification reaction solution, negative control, and positive control; And / or, the concentration ratio of the forward primer to the reverse primer in the primer set is (2-100):1; And / or, the final concentration of any primer in the primer set is 5-100 nM.

[0014] Preferably, the negative control is ddH2O, and the positive control is the internal reference GAPDH plasmid.

[0015] A fourth aspect of the present invention is to provide a microfluidic chip for the detection of multiplex bacterial and fungal nucleic acids, comprising the nucleic acid detection primer and probe set described in the first aspect; an array of fluorescent probe complexes is immobilized within the microfluidic chip; the fluorescent probe complexes are obtained by annealing Arm probes with corresponding Quencher probes and Anchor probes.

[0016] Furthermore, the microfluidic chip is a convection PCR chip.

[0017] A fifth aspect of the present invention is to provide a method for detecting multiple bacterial and fungal nucleic acids for non-diagnostic purposes, the steps of which include: S1. Obtain or provide the nucleic acid of the sample to be tested; S2. Using the extracted nucleic acid as a template, convective PCR amplification is performed in the microfluidic chip described in the fourth aspect; S3. Detect fluorescence signal; S4. Determine the results based on the amplification curves of the internal control and the target.

[0018] Furthermore, the reaction program for the convective PCR amplification is 94℃ for 15s, 58℃ for 20s, and 72℃ for 30s for a total of 1h.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention designs 47 primer pairs and Toehold chain displacement fluorescent probes for 47 bacterial and fungal pathogens. The primer pairs complete specific amplification in a single tube, and the Toehold chain displacement fluorescent probes can efficiently generate fluorescence signals by undergoing chain displacement reactions with the target. The primer pairs and Toehold chain displacement fluorescent probes can avoid the interference problem of multiple amplification. The combination in a single tube can efficiently detect any bacterial or fungal pathogens, which has significant value for the detection of environmental or clinical samples.

[0020] Compared to high-throughput sequencing, the microfluidic convection PCR amplification detection method of this invention has higher sensitivity, higher detection rate, lower cost per sample, shorter detection cycle, and lower requirements for operators and analysts. Compared with conventional quantitative PCR and tNGS detection, it is less time-consuming, the equipment is lightweight, a single tube can detect more pathogens, and the operation is simple and the cost is low. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results of amplification of 10 pathogens using the primer set designed in Example 1 of this invention are shown.

[0023] Figure 2 This is a specificity detection curve for five pathogens in Example 7 of the present invention.

[0024] Figure 3 This is a specificity detection curve of five pathogens in Example 8 of the present invention. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] This invention, through literature review and expert consensus analysis in the field of bacterial and fungal infections, summarizes the latest relevant literature and reviews, and combines this with current epidemiological characteristics of bacterial and fungal infections to select the top-ranking pathogens and determine a target list. Ultimately, 47 bacterial and fungal pathogens were identified, as shown in Table 1 below.

[0027] Table 1: List of 47 bacterial and fungal pathogens

[0028] Based on the identified pathogens, specific primers and probes are designed, including the following steps: 1. Downloaded all high-quality genome sequences of 47 bacterial, fungal pathogens, and humans from the NCBI database. Through bioinformatics alignment analysis, specific sequences conserved within the species and specific to the interspecies were selected for each pathogen. Primer design was performed using software, and the final primer pool underwent primer dimer analysis. Probes were designed using a Toehold chain substitution fluorescent probe structure, targeting conserved within the species and specific to the interspecies. The secondary structure and thermodynamic parameters were optimized using software. The Tm values ​​of all probe binding regions were kept consistent to avoid hairpins, self-dimers, and cross-dimers, ensuring compatibility for multiplex detection. Each target consisted of three oligonucleotide chains: an Anchor probe (5' end labeled with the fluorescent group Texas Red), a bridging Arm probe, and a Quencher probe (3' end labeled with the quencher group BHQ1). These three chains pre-formed a stable complex. During detection, the target sequence preferentially binds to the probe through a chain substitution reaction, displacing the fluorescently labeled chain, dequenching the fluorescent signal, and generating a specific fluorescent signal. 2. Selecting suitable primers and probes. In the primer design stage, software is used to eliminate primer dimers and non-specific amplification. Experimental screening involves diluting primers to a working solution concentration of 10 μM, extracting nucleic acids from samples, amplifying sample nucleic acids using primer pairs, and performing agarose gel electrophoresis on the amplification products to verify primer specificity. Primers that cause primer dimers or have low amplification efficiency are removed, and new primers are designed and synthesized. In the probe design stage, software analysis is used to avoid hairpins, self-dimers, and cross-dimers, ensuring compatibility with multiplex detection. Experimental functional verification of the probes involves diluting Anchor probes (5' end labeled with the fluorescent group Texas Red), bridging Arm probes, and Quencher probes (3' end labeled with the quencher group BHQ1) to a working solution concentration of 10 μM, mixing them in a specific ratio, and annealing to prepare stable 3-stranded probes. Synthesized target-specific ssDNA sequences are added to the products, and the replacement efficiency is observed using a real-time PCR instrument. Probes with poor replacement efficiency are removed, and new probes are designed and synthesized. 3. Primer and probe specificity confirmation. Corresponding plasmids were synthesized for the target sequences of 47 bacterial and fungal pathogens. The plasmids were quantified using digital PCR, quality control samples were prepared, and primer and probe specificity was tested. 4. Internal control design. Forward and reverse primers and probes were designed using the GAPDH gene as an internal control.

[0029] The final primer set with nucleotide sequences as shown in SEQ ID NO. 1-96 and the Toehold chain substitution fluorescent probe set as shown in SEQ ID NO. 97-192 were designed. The Toehold chain substitution fluorescent probe set includes an Arm probe, a Quencher probe, and an Anchor probe. The Arm probe and the Quencher probe correspond to the odd and even sequences in the nucleotide sequences shown in SEQ ID NO. 97-192, respectively, and the nucleotide sequence of the Anchor probe is shown in SEQ ID NO. 193. See Tables 2 and 3 for details.

[0030] The primer sets mentioned above have minimal interference with each other and do not produce dimers, enabling specific amplification in a single tube. The Toehold chain displacement fluorescent probe can efficiently undergo a chain displacement reaction with the target to generate a fluorescent signal. The primer pairs and the Toehold chain displacement fluorescent probe can avoid the interference problem of multiplex amplification. The binding in a single tube can efficiently detect any bacterial or fungal pathogens.

[0031] 5. Primer pool preparation. Different primers have different amplification efficiencies. To balance the amplification efficiencies among primers as much as possible, different primer concentration ratios are tested. The target product needs to be replaced with the probe. The forward and reverse primers need to be prepared according to the asymmetric PCR concentration ratio. Preferably, the concentration ratio of the forward primer to the reverse primer is 100:1, and the final primer concentration is 50 nM.

[0032] 6. Probe Spotting. After the above probes have passed functional verification, stable 3-chain probes will be prepared and spotted onto the microfluidic chip.

[0033] 7. PCR reaction system. Based on the characteristics of primer and probe hybridization substitution, an amplification buffer with no exonuclease activity and compatible with multiplex amplification should be selected.

[0034] 8. Optimization of PCR reaction conditions. Based on primer characteristics and testing at different annealing temperatures, a suitable annealing temperature was selected.

[0035] 9. Set up quality control: the negative control is ddH2O, and the positive control is the internal reference GAPDH plasmid.

[0036] Combining the primer-probe set and microfluidic chip designed above, the multiplex bacterial and fungal detection method of this invention is as follows: 1) Extract nucleic acid from the sample to be tested, with a nucleic acid concentration ≥20 ng / uL; 2) Microfluidic convection PCR detection: using extracted nucleic acid as a template, PCR amplification is performed in a microfluidic chip using a primer pool, and the amplification signal is detected. 3) Result Interpretation: After amplification, observe the amplification curve. The horizontal axis represents amplification time. If the internal reference GAPDH gene amplifies normally within 1 hour and peaks within <30 minutes, and peaks appear in the bacterial and fungal tests within <30 minutes, the result is considered positive. If the internal reference GAPDH gene amplifies normally and peaks within <30 minutes, and no peaks appear in the bacterial and fungal tests, the result is considered negative. If the internal reference GAPDH gene amplification is abnormal, the result is invalid and needs to be repeated.

[0037] The methods described above can efficiently detect one or more bacterial or fungal pathogens. It is understood that a positive or negative test result objectively represents the presence or absence of pathogens in the sample. The samples in question include, but are not limited to, environmental or clinical samples. Environmental samples include, but are not limited to, other samples taken outside of human bodies.

[0038] It is understandable that the above detection methods can be used for clinical diagnosis or auxiliary diagnosis, as well as for non-diagnostic detection of environmental samples. The test results only indicate whether the pathogens are present or absent in the environmental samples.

[0039] Example 1: Design and validation of specific primers and probes for detecting 47 bacterial and fungal pathogens.

[0040] 1. Primer pool design: Download all high-quality genome sequences of 47 bacterial, fungal pathogens and humans from the NCBI database. Through bioinformatics comparison analysis, select species-conserved and species-specific sequences for each pathogen. Primer design was performed using Primer 5.0. The designed primers maintained a uniform Tm value (60℃±5℃), amplicon length of 100-300 bp, primer length of 15-25 bp, and GC content of 40-60%. The final primer pool required primer dimer analysis.

[0041] 2. Probe Design: The probes employ a Toehold chain displacement fluorescent probe structure design, targeting species-conserved and species-specific target sequences. Secondary structures and thermodynamic parameters are optimized using software such as NUPACK and ViennaRNA. The Tm values ​​of all probe binding regions are kept consistent to avoid hairpins, self-dimers, and cross-dimers, ensuring compatibility for multiplex detection. Each target consists of three oligonucleotide chains: an anchor probe (5' end labeled with the fluorescent group Texas Red), a bridging Arm probe, and a Quencher probe (3' end labeled with the quencher group BHQ1). These three chains pre-form a stable complex. During detection, the target sequence preferentially binds to the probe via a chain displacement reaction, displacing the fluorescently labeled chain, dequenching the fluorescent chain, and generating a specific fluorescent signal.

[0042] 3. Experimental Validation: By purchasing ATCC standard strains of microorganisms, standard nucleic acids, constructing standard quality plasmids or clinical positive samples, all primers in the primer pool were amplified and run on gels. Primers that did not amplify bands or produced cross-reactions were removed, and new primers were designed to supplement the primer pool and then experimentally validated.

[0043] 4. Functional validation was performed on specific probes designed for 47 bacterial and fungal pathogens. Each target consisted of three oligonucleotide chains: an anchor probe (5' end labeled with a fluorescent group), a bridging Arm probe, and a Quencher probe (3' end labeled with a quencher group). The three probes were formulated as single-anchor probes, 2-strand probes, and 3-strand probes, respectively. The 2-strand and 3-strand probes were mixed in specific ratios, with a preferred 2-strand probe ratio of 1:2 for annealing to prepare a stable 2-strand probe, and a preferred 3-strand probe ratio of 1:2:3 for annealing to prepare a stable 3-strand probe. Synthetic target-specific ssDNA sequences were added to the annealing products of the 3-strand probes. The substitution efficiency was observed using a quantitative real-time PCR instrument. The relative fluorescence intensity ratio of each probe complex was calculated using the fluorescence intensity of the single anchor probe as a normalization benchmark (set to 100). A qualified probe should meet the following requirements: the relative fluorescence intensity of a 2-strand probe is 100±10, the relative fluorescence intensity of a 3-strand probe is 10±5, the fluorescence intensity recovers to above 50 after the addition of target ssDNA, and the ratio of 3-strand + ssDNA to 2-strand is ≥50%. Probes with poor substitution efficiency should be removed and new probes should be designed and synthesized.

[0044] The final primer sequences designed for 47 bacterial and fungal pathogens are shown in Table 2.

[0045] Table 2: Primers for 47 bacterial, fungal pathogens and humans

[0046] The probe sequences designed for 47 bacterial and fungal pathogens are shown in Table 3.

[0047] Table 3: Probe sets for 47 bacterial, fungal pathogens and humans

[0048] Special note: When the sequences in the nucleotide sequence table provided by this invention are inconsistent with those in Tables 3 and 4, the sequences shown in Tables 3 and 4 in this specification shall prevail.

[0049] Primers for 47 bacterial and fungal pathogens were amplified using gel electrophoresis, and all 47 primer pairs amplified normally. Results for 10 pathogens are presented as examples. Figure 1 The target bands are amplified, numbered 1-10, corresponding to Acinetobacter baumannii, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Chlamydia pneumoniae, respectively.

[0050] Functional validation of specific probes designed for 47 bacterial and fungal pathogens revealed that all probes met the design requirements. Results for 10 pathogens are presented as examples in Table 4 below.

[0051] Table 4: Functional validation results of 10 pathogen probes

[0052] Example 2: Primer combinations for detecting 47 bacterial and fungal pathogens

[0053] Different primers have different amplification efficiencies. To balance the amplification efficiencies among primers as much as possible, different primer concentration ratios were tested. The target product needs to be replaced with the probe. The forward and reverse primers need to be mixed according to asymmetric PCR concentrations, with a forward primer:reverse primer ratio of 100:1-2:1, preferably 100:1. The final primer concentration is 5-100 nM, preferably 50 nM. The target detection primer set shown in Table 2 was obtained through the development process in Example 1. All primer powders were dissolved to form a high-concentration primer mixture, and primer pool T47P was prepared in one tube.

[0054] Example 3: Spotting of probes for 47 bacterial and fungal pathogens

[0055] The Toehold chain-displacement fluorescent probes verified in Table 3 were immobilized in the form of a three-strand complex on the inner surface of the reaction chamber of the microfluidic chip, forming a probe array. The surface of the probe array region in the PDMS channel layer within the microfluidic chip was treated with oxygen plasma and modified with amino groups. The probes were covalently fixed to the surface-modified chip substrate. Probes of different species were spotted according to a preset spatial arrangement, with probe spot diameters of 150±20 μm and center-to-center spacing of ≥300 μm. Probes of different species were spotted at different positions on the chip. The amplified targets hybridized with the probes at the corresponding positions to generate fluorescent signals. The spatial location of the fluorescent signals was identified by image analysis to determine the pathogen. There were 47 targets + 1 internal control = 48 spots, with each spot corresponding to one species.

[0056] Example 4: Composition of a detection kit for 47 bacterial and fungal pathogens

[0057] The detection kits for 47 bacterial and fungal pathogens include an exonuclease-free and multiplex-compatible amplification buffer (2×AmpExcellent™ Multiplex PCR Mix, sourced from Kangwei Century), a T47P primer pool, a negative control (ddH2O), and a positive control (internal reference GAPDH plasmid). Details are shown in the table below.

[0058] Example 5: Detection methods for 47 bacterial and fungal pathogens

[0059] 1) Sample pretreatment and nucleic acid extraction

[0060] Take 200 μl of the sample to be tested. Any commercially available nucleic acid extraction kit can be used. Please refer to the corresponding instruction manual for specific extraction methods.

[0061] 2) Microfluidic convection PCR amplification detection

[0062] Using extracted nucleic acid as a template, PCR amplification was performed in a microfluidic chip using a primer pool (this microfluidic chip is a self-developed product of Shanghai Tuoruis Diagnostic Technology Co., Ltd., with patent publication number CN120944682A), and the amplification signal was detected.

[0063] The reaction system for convection PCR amplification was as follows: 10 μL of 2×AmpExcellent™ Multiplex PCR Mix, 5 μL of T47P primer pool, and 5 μL of DNA, for a total volume of 20 μL. The amplification program for this kit was: 94℃ for 15 s, 58℃ for 20 s, and 72℃ for 30 s for a total of 1 h; fluorescence signals were collected by taking pictures at 58℃.

[0064] 3) Result Interpretation

[0065] Interpretation of results: After amplification, observe the amplification curve. The horizontal axis represents amplification time. If the internal reference GAPDH gene amplifies normally within 1 hour and peaks within <30 minutes, and peaks appear in each bacterial and fungal test within <30 minutes, the result is considered positive. If the internal reference GAPDH gene amplifies normally and peaks within <30 minutes, and no peaks appear in each bacterial and fungal test, the result is considered negative. If the internal reference GAPDH gene amplification is abnormal, the result is invalid and must be repeated.

[0066] Example 6: Single-layer validation of primers and probes for 47 bacterial and fungal pathogens.

[0067] Using target plasmids of 47 pathogens as templates (concentration 10^4 copies / mL), singlet fluorescent PCR was performed on a microarray using corresponding specific primers and probes. Internal control GAPDH primers were added for each reaction. Results showed that specific amplification curves were observed for all 47 targets, with detection values ​​ranging from 26.0 to 31.0. The internal control showed normal peaks, while the negative control showed no amplification. Detailed results are shown in Table 5. Table 5: Single-pass validation results of primers and probes for 47 bacterial and fungal pathogens

[0068] Example 7: Validation of the discriminative power of multiple detection.

[0069] Equal amounts of target plasmids for 47 pathogens (10^4 copies / mL each) were mixed and used as templates. Single-tube detection was performed using the primer and probe pools described in this invention. Results showed that specific amplification curves were observed for all 47 targets, and the detection values ​​for each target were consistent with the single-tube detection results (difference ≤1.0). Internal controls showed normal peaks, and the negative control showed no amplification, indicating no interference between the primers / probes. Specific results are shown in Table 6. Figure 2 (Five target results are shown as examples. The horizontal axis represents amplification time, and the vertical axis represents fluorescence value.)

[0070] Table 6: Results of the discrimination capability verification of multiple detection methods

[0071] Further cross-reactivity verification was conducted: cross-reactivity tests were performed on the target probe primers for pathogens within and between genera, respectively. No specific amplification signal (no peak) was found, indicating that the primer-probe combination described in this invention has good specificity, and there was no cross-reactivity among the 47 targets. Representative cross-reactivity verification results within and between genera are shown in Tables 7 and 8.

[0072] Table 7: Results of cross-reactivity validation within representative genera

[0073] Table 8: Results of Cross-Reactivity Validation Between Species

[0074] Note: Other bacterial mixed templates: Plasmids of 37 bacterial targets other than the target to be tested were mixed in equal amounts (10^4 copies / mL each).

[0075] Fungal mixed template: Equal amounts of 8 fungal target plasmids were mixed (10^4 copies / mL each).

[0076] Human genomic DNA: concentration 10 ng / μL.

[0077] Example 8: LOD detection of the five pathogens included using the above method.

[0078] Five pathogens were serially diluted (10^5 copies / mL, 10^4 copies / mL, 10^3 copies / mL), with each dilution performed in triplicate. The results are shown in Table 9 below. Figure 3 (The result of 10^5 copies / mL is shown as an example.)

[0079] Table 9: LOD Detection Results of 5 Pathogens

[0080] The results showed that microfluidic convection PCR had good detection performance, and all targets could be stably detected in the range of 10^3 to 10^5 copies / mL.

[0081] Example 9: Clinical Sample Validation

[0082] To verify the performance of the detection of 47 bacterial and fungal pathogens provided by this invention, the same clinical sample with a positive tNGS result was selected and subjected to microfluidic convection PCR detection. The difference between the two results was compared, and the results are shown in Table 10 below.

[0083] Table 10: Comparison of microfluidic convection PCR detection results with high-throughput sequencing (tNGS) results

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer and probe set for the detection of multiplex bacterial and fungal nucleic acids, characterized in that, The kit includes a primer set with nucleotide sequences as shown in SEQ ID NO. 1-94, and a Toehold chain substitution fluorescent probe set. The Toehold chain substitution fluorescent probe set includes an Arm probe, a Quencher probe, and an Anchor probe. The Arm probe and the Quencher probe correspond to the odd and even sequences in the nucleotide sequences shown in SEQ ID NO. 97-190, respectively, and the nucleotide sequence of the Anchor probe is shown in SEQ ID NO.

193.

2. The nucleic acid detection primer and probe set as described in claim 1, characterized in that, It also includes internal reference gene detection primer pairs and fluorescent probe sets.

3. The nucleic acid detection primer and probe set as described in claim 1, characterized in that, The primer set also includes internal reference gene detection primer pairs with nucleotide sequences as shown in SEQ ID NO. 95-96, and the Toehold chain substitution fluorescent probe set also includes internal reference Arm probe and internal reference Quencher probe with nucleotide sequences as shown in SEQ ID NO. 191 and 192, respectively.

4. The use of the nucleic acid detection primer and probe set according to any one of claims 1-3 in the preparation of multiplex bacterial and fungal nucleic acid detection products.

5. A multiplex bacterial and fungal nucleic acid detection kit, characterized in that, Includes the nucleic acid detection primer and probe set as described in any one of claims 1-3.

6. The detection kit as described in claim 5, characterized in that, The test kit also includes at least one of the following: nucleic acid extraction reagent, amplification reaction solution, negative control, and positive control. And / or, the concentration ratio of the forward primer to the reverse primer in the primer set is (2-100):1; And / or, the final concentration of any primer in the primer set is 5-100 nM.

7. A microfluidic chip for detecting multiplex bacterial and fungal nucleic acids, characterized in that, The invention includes the nucleic acid detection primer and probe set according to any one of claims 1-3; the microfluidic chip contains an array of fluorescent probe complexes; the fluorescent probe complexes are obtained by annealing Arm probes with corresponding Quencher probes and Anchor probes.

8. The microfluidic chip as described in claim 7, characterized in that, The microfluidic chip is a convection PCR chip.

9. A method for detecting multiple bacterial and fungal nucleic acids for non-diagnostic purposes, characterized in that, include: S1. Obtain or provide the nucleic acid of the sample to be tested; S2. Using the extracted nucleic acid as a template, perform convection PCR amplification in the microfluidic chip described in claim 7 or 8; S3. Detect fluorescence signal; S4. Determine the results based on the amplification curves of the internal control and the target.

10. The method as described in claim 9, characterized in that, The reaction program for the convective PCR amplification was 94℃ for 15s, 58℃ for 20s, and 72℃ for 30s for a total of 1h.

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