A kit for rapidly detecting HAP / VAP pathogenic bacteria and drug resistance genes and application thereof

Abstract

This invention discloses a rapid detection kit for HAP / VAP pathogens and drug resistance genes, and its applications, belonging to the field of biomedical detection technology. Addressing the problems of long detection processes, complex operations, and poor sample adaptability in existing tNGS technology, this invention provides an optimized detection kit. The kit includes: a sample pretreatment and nucleic acid extraction reagent system, whose sample preservation solution contains 5% dithiothreitol and 2.5% potassium azide for simultaneous sample liquefaction and salt ion chelation during transportation; and an amplification and library construction reagent system, which contains a primer mixture consisting of 175 primer pairs, E. coli synthase III, and separately added magnesium ions. By integrating the above reagent systems and optimizing the entire process, this invention reduces the total time from sample pretreatment to report generation to less than 2.5 hours. This invention has the advantages of extremely fast detection speed, wide sample applicability, high operational stability, and ease of automation.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and more specifically, to a kit for rapid detection of pathogens and related drug resistance genes in hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP), and its application. Background Technology

[0002] Hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) are common and serious infectious diseases in clinical practice, characterized by rapid onset, rapid progression, complex pathogens, and high drug resistance rates. Dozens of pathogens and more than ten drug-resistant genes are associated with HAP / VAP infections. Rapid and accurate identification of the causative bacteria and their drug-resistant genes is crucial for guiding precise clinical medication and improving patient prognosis.

[0003] Currently, there are two main types of detection technologies commonly used in clinical practice: one is quantitative real-time PCR amplification technology, and the other is targeted multiplex PCR technology (tNGS) based on high-throughput sequencing.

[0004] Quantitative real-time PCR (qPCR) technology has the advantages of fast detection speed and convenient operation, typically completing amplification within 3 hours. However, this technology has significant drawbacks: First, each tube can only detect a few pathogens; to detect dozens of microorganisms, dozens of primers need to be prepared and premixed separately, making the operation cumbersome and resulting in low throughput. Second, the false positive problem caused by amplification of a single target region is quite serious. Third, it cannot effectively genotype polymorphic random mutations in drug resistance genes.

[0005] tNGS technology has advantages such as low cost, wide detection coverage, and high specificity, but existing technical solutions still have many problems: First, the experimental operation time is long. From sample pretreatment, nucleic acid extraction, amplification, library construction to sequencing, it usually takes 15-24 hours. Even with nanopore sequencing technology, the whole process takes more than 8 hours. For patients with highly drug-resistant HAP / VAP infections, especially critically ill patients in the ICU, this testing time will miss the golden treatment period. Second, the enzymes used in tNGS are relatively special. Hundreds of primer pairs react in the same tube, which requires extremely high requirements for the amplification environment. Respiratory tract-related samples are complex (sputum, lavage fluid, swabs, etc.), and the pretreatment operation procedures for different samples vary greatly, making standardization difficult. Third, the experimental steps are cumbersome, involving multiple tube transfers, purification, and volume adjustment operations. The PCR reaction system is basically 10-20 μl, which is too small to achieve accurate full automation.

[0006] Therefore, developing a detection kit and method for HAP / VAP pathogens and drug resistance genes that can balance detection speed, sensitivity, stability, and automation adaptability is of significant clinical value and urgently needed. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a kit and detection method for rapid detection of HAP / VAP pathogens and drug resistance genes, so as to complete the entire detection process from sample pretreatment to report issuance within 2.5 hours, while improving the stability, applicability and automation adaptability of the detection.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a kit for detecting HAP / VAP pathogens and drug resistance genes, the kit comprising: A sample pretreatment and nucleic acid extraction reagent system includes a sample preservation solution containing 5% guanidine hydrochloride, EDTA, 5% dithiothreitol solution and 2.5% potassium azide solution. as well as, The amplification and library preparation reagent system includes: (a) Primer mixture, which consists of 175 pairs of primers remaining after removing degenerate primers from the primers for detecting HAP / VAP pathogens and related drug resistance genes as described in patent ZL 2023 1 0714764.6, and the concentration of each primer is adjusted to 100-250 nM. (b) Escherichia coli synthase III as an amplification enzyme; (c) Magnesium ions added separately.

[0009] Furthermore, in the sample pretreatment and nucleic acid extraction reagent system, the sample preservation solution is used to simultaneously liquefy the sample during sample transportation.

[0010] Furthermore, the pathogenic bacteria detected by the kit include: Escherichia coli, Nocardia anthracis, Staphylococcus aureus, Klebsiella pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptococcus pneumoniae, Haemophilus influenzae, Legionella, Streptococcus constellations, Streptococcus pyogenes, Candida albicans, Candida tropicalis, Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, Aspergillus flavus, and Pneumocystis jirovecii; the drug resistance genes detected include: MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, MCR-8, MCR-9, MCR-10, TEM, blaCTX-M, blaGES, blaIMP, blaOXA-23, blaVIM, embB, mecA, vanA, and vanB.

[0011] Furthermore, in the amplification library preparation reagent system, the Escherichia coli synthase III is sensitive to the ion concentration in the PCR reaction system, and the separately added magnesium ions are used to optimize the amplification efficiency.

[0012] Furthermore, the kit is used for the detection of HAP / VAP infection-related samples, which are selected from sputum, lavage fluid, or swabs.

[0013] Secondly, the present invention provides a detection method using the above-mentioned reagent kit, the method comprising the following steps: Step 1: The sample is processed using the sample pretreatment and nucleic acid extraction reagent system, wherein the sample is liquefied and salt ion chelated in a preservation solution containing dithiothreitol and potassium azide, and then nucleic acid is extracted using a fully automated nucleic acid extractor. Step 2: Use the described amplification and library construction reagent system to amplify and construct a library from the extracted nucleic acid; Step 3: Perform sequencing analysis; The entire process, from sample pretreatment to report issuance, takes less than 2.5 hours.

[0014] Furthermore, the fully automated nucleic acid extractor completes nucleic acid extraction in 15 minutes.

[0015] Furthermore, in the amplification library preparation reagent system, after adjusting the primer concentration to 100-250 nM, the library concentration was increased without increasing the primer dimer concentration.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly faster detection speed: This invention optimizes the sample pretreatment system (DTT for rapid liquefaction and potassium azide chelate ions), simplifies the primer system (175 pairs, removing degenerate primers), replaces high-fidelity E. coli synthase III and optimizes the magnesium ion addition method, and integrates and compresses the entire process, so that the entire process time from sample pretreatment to report issuance is shortened to less than 2.5 hours. Compared with the traditional tNGS technology (8-24 hours), it has achieved a breakthrough speed-up, which has secured a golden treatment window for critically ill patients.

[0017] (2) Significantly improved applicability and stability: This invention adds dithiothreitol (DTT) and potassium azide to the sample preservation solution. DTT can rapidly liquefy highly viscous sputum and lavage fluid without damaging microorganisms; potassium azide can strongly chelate high concentrations of salt ions in the lavage fluid, reducing the salt ion removal pressure during nucleic acid extraction and providing a good enzyme reaction environment for subsequent amplification. This pretreatment system is applicable to various sample types such as sputum, lavage fluid, and swabs, and the operation process is consistent, making it easy to achieve mechanized operation and laying a good foundation for full-process automation.

[0018] (3) Improved amplification efficiency and library concentration: Based on the primer system of the prior patent, this invention removes degenerate primers, reduces the number of primers to 175 pairs, and adjusts the primer concentration from 50 nM to 100-250 nM. Under the same PCR conditions, primer dimers did not show significant changes, while library concentration was significantly improved. At the same time, the low-fidelity PCR amplification enzyme was replaced with E. coli synthase III, which has excellent amplification efficiency. Combined with the precise addition of magnesium ions, the amplification efficiency and detection sensitivity were further improved.

[0019] (4) Balancing detection sensitivity and specificity: While ensuring detection speed, this invention achieves efficient detection of 12 bacteria, 7 fungi and 19 drug resistance genes through optimized primer design and enzyme system. It has the characteristics of wide coverage and high specificity, effectively solving the problems of high false positive rate and inability to genotype drug resistance gene mutations in traditional quantitative PCR. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a graph showing the fragmentation analysis results before primer adjustment, with the PCR reaction conditions and experimental conditions remaining unchanged.

[0022] Figure 2 This is a graph showing the fragmentation analysis results after primer adjustment, with the PCR reaction conditions and experimental conditions remaining unchanged. Detailed Implementation

[0023] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Experimental methods not specifically described in the following embodiments were performed according to conventional methods and conditions, or as selected in the product manual. All software used in these embodiments is commercially available software or open-source free software.

[0024] Example 1: Composition and Preparation of the Reagent Kit This embodiment provides a kit for rapid detection of HAP / VAP pathogens and drug resistance genes, with the following specific components: 1. Sample pretreatment and nucleic acid extraction reagent system Sample preservation solution: Prepare a mixed solution containing 5% (w / v) guanidine hydrochloride, 5% (w / v) dithiothreitol (DTT), 2.5% (w / v) potassium azide (KN3) and EDTA, adjust the pH to 7.0-7.5, filter aseptically and dispense.

[0025] Nucleic acid extraction reagent: SDK60120 extraction reagent is used, which is compatible with SSNP9600A fully automated nucleic acid extractor.

[0026] 2. Amplification and Library Construction Reagent System Primer mixture: Based on the primer sequences disclosed in patent ZL 2023 1 0714764.6, all degenerate primers were removed, retaining 175 pairs of specific primers, targeting 12 bacteria, 7 fungi, and 19 drug resistance genes, respectively. The concentration of each primer was adjusted to 100-250 nM, and the mixture was then aliquoted.

[0027] Amplification enzyme: Escherichia coli synthase III (pol).

[0028] PCR reaction buffer: contains Tris-HCl, KCl, dNTPs, etc. Magnesium ions are packaged separately and added separately at the optimized concentration when used.

[0029] Example 2: Detection Method and Full-Process Time Validation The specific steps for testing clinical samples using the kit of this invention are as follows: 1. Sample pretreatment and nucleic acid extraction Collect sputum, irrigation fluid, or swab samples and add them directly to a sample preservation solution containing DTT and potassium azide. The sample liquefaction and salt ion chelation are completed simultaneously during transportation (approximately 30 minutes).

[0030] Once the samples arrived at the laboratory, they were directly transferred to the SSNP9600A fully automated nucleic acid extractor and extracted using the SDK60120 extraction reagent. The extraction program was set to 15 minutes.

[0031] The total time for this step is approximately 45 minutes (including 30 minutes for transport and liquefaction + 15 minutes for extraction).

[0032] 2. Library construction and amplification Take 5 μL of the extracted nucleic acid and add it to a PCR reaction system containing a mixture of 175 primer pairs (100-250 nM), E. coli synthase III (2 U), magnesium ions (2.5 mM) and reaction buffer, for a total volume of 20 μL.

[0033] Amplification program: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 20 s, 60℃ annealing for 30 s, 72℃ extension for 40 s, for a total of 25 cycles; 72℃ final extension for 5 min.

[0034] The amplified products do not require purification and can be directly used for library construction steps such as end repair and adapter ligation. The total library construction time is about 45 minutes.

[0035] The total time for this step is approximately 60 minutes.

[0036] 3. Sequencing analysis The constructed library was subjected to high-throughput sequencing (using a benchtop sequencer), which took approximately 45 minutes.

[0037] Sequencing data is automatically analyzed using bioinformatics, compared against pathogen and drug resistance gene databases, and reports are generated.

[0038] The total time for this step is approximately 45 minutes.

[0039] 4. Total time for the entire process: Sample transportation and liquefaction 30 minutes + nucleic acid extraction 15 minutes + amplification and library preparation 60 minutes + sequencing analysis 45 minutes = 150 minutes (2.5 hours).

[0040] Example 3: Verification of the effect of primer system adjustment To verify the effect of primer system adjustment, a comparative experiment was set up: Control group: The original primer system in patent ZL 2023 1 0714764.6 (including degenerate primers, a total of about 220 primer pairs, and a concentration of 50 nM for each primer) was used.

[0041] Experimental group: After removing degenerate primers, 175 primer pairs remained, and the concentration of each primer was adjusted to 100-250 nM. Taking the detection of Pseudomonas aeruginosa as an example, Table 1 shows the primer sequences and reference concentrations for Pseudomonas aeruginosa.

[0042] Table 1 Primer sequences and reference concentrations for Pseudomonas aeruginosa Under the same PCR conditions (E. coli synthase III, magnesium ions 2.5 mM), the same positive reference sample was amplified and a library was constructed. The library concentration and primer dimer formation were then measured.

[0043] The results are as follows Figure 1 and Figure 2 As shown in Table 2, the library concentration was significantly increased after primer adjustment.

[0044] Table 2 Comparison of library concentrations before and after primer adjustment Example 4: Validation of the effectiveness of the sample pretreatment system The same bronchoalveolar lavage fluid was collected at the same time, with the same experimenter and experimental site, the same downstream operation procedure, and the same amount of sequencing data. The detection of samples mixed with DTT premixed solution and samples without premixed solution was analyzed. The results are shown in Tables 3 and 4. The detection effect of samples mixed with DTT premixed solution was better.

[0045] Table 3. Detection of samples without DTT premixed solution Table 4. Detection of DTT premixed solution in samples Example 5: Verification of the effect of adding potassium azide solution to the premixed solution Adding potassium azide (KN3) solution to the premix can strongly chelate the high concentration of salt ions in the washing solution, reducing the pressure of salt ion removal during nucleic acid extraction and providing a favorable environment for enzyme operation in amplification.

[0046] This invention used 11 samples with a 260 / 230 ratio less than 1.0 that failed to construct a library using tNGS in our laboratory. The selected samples were re-extracted and reconstructed using premixed solutions with and without KN3. As shown in Table 5, the library concentrations after KN3 were all higher than those without KN3.

[0047] Table 5. Analysis of the effect of adding potassium azide solution to the premixed solution. Finally, it should be noted that the above description of the present invention is merely a preferred embodiment and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. 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 kit for detecting HAP / VAP pathogens and drug resistance genes, characterized in that, The kit includes: A sample pretreatment and nucleic acid extraction reagent system includes a sample preservation solution containing 5% guanidine hydrochloride, EDTA, 5% dithiothreitol solution and 2.5% potassium azide solution. as well as, The amplification and library preparation reagent system includes: (a) Primer mixture, which consists of 175 pairs of primers remaining after removing degenerate primers from the primers for detecting HAP / VAP pathogens and related drug resistance genes as described in patent ZL 2023 1 0714764.6, and the concentration of each primer is adjusted to 100-250 nM. (b) Escherichia coli synthase III as an amplification enzyme; (c) Magnesium ions added separately.

2. The reagent kit according to claim 1, characterized in that, In the sample pretreatment and nucleic acid extraction reagent system, the sample preservation solution is used to simultaneously liquefy the sample during sample transportation.

3. The reagent kit according to claim 1, characterized in that, The pathogenic bacteria include: Escherichia coli, Nocardia anthracis, Staphylococcus aureus, Klebsiella pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptococcus pneumoniae, Haemophilus influenzae, Legionella, Streptococcus constellationus, Streptococcus pyogenes, Candida albicans, Candida tropicalis, Aspergillus niger, Aspergillus fumigatus, Aspergillus terreus, Aspergillus flavus, and Pneumocystis jirovecii; The drug resistance genes include: MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, MCR-8, MCR-9, MCR-10, TEM, blaCTX-M, blaGES, blaIMP, blaOXA-23, blaVIM, embB, mecA, vanA, and vanB.

4. The reagent kit according to claim 1, characterized in that, In the amplification and library preparation reagent system, the Escherichia coli synthase III is sensitive to the ion concentration in the PCR reaction system, and the amplification efficiency is optimized by adding magnesium ions separately.

5. The reagent kit according to claim 1, characterized in that, The kit is used for the detection of HAP / VAP infection-related samples, which are selected from sputum, lavage fluid or swabs.

6. A detection method using the kit according to any one of claims 1-5, characterized in that, The method includes the following steps: Step 1: The sample is processed using the sample pretreatment and nucleic acid extraction reagent system, wherein the sample is liquefied and salt ion chelated in a preservation solution containing dithiothreitol and potassium azide, and then nucleic acid is extracted using a fully automated nucleic acid extractor. Step 2: Use the described amplification and library construction reagent system to amplify and construct a library from the extracted nucleic acid; Step 3: Perform sequencing analysis; The entire process, from sample pretreatment to report issuance, takes less than 2.5 hours.

7. The detection method according to claim 6, characterized in that, The fully automated nucleic acid extractor completes nucleic acid extraction in 15 minutes.

8. The detection method according to claim 6, characterized in that, In the amplification and library preparation reagent system, after adjusting the primer concentration to 100-250 nM, the library concentration was increased without increasing the primer dimer concentration.

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