A primer probe composition for gastrointestinal pathogen detection and application thereof

Abstract

The application relates to a primer probe composition and application for gastrointestinal pathogen detection. The primer probe composition comprises composition 1 and composition 2; the composition 1 comprises one universal label primer and 22 groups of primer probe groups respectively used for detection of 15 gastrointestinal pathogens to be detected; the composition 2 comprises one universal label primer and 17 groups of primer probe groups respectively used for detection of other 17 gastrointestinal pathogens to be detected; wherein each group of the primer probe group comprises one upstream primer, one downstream primer and one probe, and the 5' end of the upstream primer and the downstream primer is connected with the nucleotide sequence of the universal label primer, and the nucleotide sequence of the universal label primer is shown in SEQ ID NO: 121. The primer probe composition or the kit comprising the primer probe composition can realize simultaneous detection of 32 gastrointestinal pathogens.

Description

Technical Field

[0001] This application relates to the field of molecular biology technology, and in particular to a primer-probe composition for the detection of gastrointestinal pathogens and its application. Background Technology

[0002] Diarrhea is a common infectious and prevalent disease in humans, especially children, the elderly, and immunocompromised individuals, ranking second only to respiratory diseases in the causes of death among infants and young children. The causes of infectious diarrhea are complex, and it can be caused by viruses, bacteria, and parasites. While different types of pathogens may present with similar symptoms, their treatment methods and outcomes vary. Therefore, from a clinical perspective, it is essential to conduct parallel screening and testing for gastrointestinal pathogens to accurately identify the specific pathogen causing the infection, facilitating timely and effective subsequent control.

[0003] From a policy perspective, in May 2019, the National Healthcare Security Administration held a video conference to launch the national pilot program for Diagnosis Related Groups (DRG) payment. The conference announced a list of 30 pilot cities for DRG payment and introduced the preparations and main content of the national pilot program. The nationwide implementation of DRG payment is inevitable. By establishing unified fixed-rate payment standards for disease diagnosis classifications, it achieves standardized utilization of medical resources and facilitates cost control. DRG (Diagnosis Related Groups) involves comprehensively analyzing a patient's condition and grouping them into different diagnostic groups for bundled treatment, thus standardizing the treatment process and controlling treatment costs. In short, it represents a shift in healthcare payment methods from "fee-for-service" to "payment-for-disease."

[0004] A syndrome refers to a set of clinical features, symptoms, or phenomena that frequently occur simultaneously in the course of certain diseases, even when the actual pathogen is unknown. Therefore, to confirm the cause, physicians typically combine tests for multiple pathogens or conduct step-by-step investigations. Under the DRG (Diagnosis Related Groups) framework, the accumulated high costs limit the combined use of multiple tests; while step-by-step investigations would prolong the treatment period and contradict the original intention of DRG implementation.

[0005] Syndrome multifactorial testing combines pathogens causing the same or similar clinical symptoms into a single detection unit. A single test yields results for all related pathogens, eliminating the need to wait for individual pathogen screening results and avoiding the cumulative costs of multiple simultaneous tests. This provides a direct and effective solution that simultaneously meets clinical diagnostic and cost-control needs. Based on the treatment requirements for gastrointestinal pathogen infections and national policy guidance, there is an urgent need to establish a rapid and efficient diagnostic method capable of simultaneously detecting multiple common diarrheal pathogens.

[0006] Currently, the main methods used in clinical pathogen detection include culture and isolation identification, immunological detection, and nucleic acid detection. Among these, isolation and culture require a long time, which does not meet the treatment needs of acute infections. Convenient and rapid immunological detection is currently the main method for detecting single pathogens, but its sensitivity is low, making it unsuitable for early diagnosis, and cross-reactivity between antigens and antibodies can easily lead to false positives. Besides operational and inherent limitations, the above two methods are also unsuitable for multiplex detection of multiple pathogens. Compared to these two methods, nucleic acid detection not only avoids false positives due to the specificity of primers and probes, but also allows for the multiplex detection of a wider range of pathogens using gene chips and multiplex melting curve methods. Therefore, it has a significant technological advantage in the detection of gastrointestinal infection syndromes. Compared to chip methods, multiplex melting curve methods still suffer from the limitation of multiplicity inherent in real-time fluorescence PCR, limiting the potential for improvement. It still requires well splitting to achieve higher multiplicity detection, thus the increased cost due to well splitting remains unavoidable. Due to its limited sensitivity, the existing gene chip technology is mainly used for screening where sensitivity requirements are not high, and its application in pathogen detection, which requires higher sensitivity, is relatively limited.

[0007] Microfluidics integrates the basic operational units of biological, chemical, and medical analysis processes—such as sample preparation, reaction, separation, and detection—onto a single micrometer-scale chip, automating the entire analytical process. With advantages such as compact size, small sample and reagent volumes, rapid reaction speed, large-scale parallel processing, and disposable nature, microfluidic chips hold immense potential in fields such as biology, chemistry, and medicine.

[0008] Biofire's FilmArray system is a representative product in the field of molecular diagnostics for syndrome multifactorial assays. It obtained FDA (Food and Drug Administration) certification as early as 2011 and can detect multiple pathogens that can cause gastrointestinal infections. However, its high cost limits its application in China, and it has not yet been able to achieve widespread adoption through NMPA (National Medical Products Administration) registration. Currently, among the gastrointestinal pathogen multifactorial assays that have obtained NMPA certification, the types of pathogens they detect are limited, mainly focusing on norovirus, rotavirus A, enteric adenovirus 40 / 41, and Salmonella and Shigella. Examples include the group A rotavirus and group F enteric adenovirus 40 / 41 nucleic acid detection kit (PCR-fluorescent probe method) represented by Chongqing Wokang Biotechnology Co., Ltd., the norovirus nucleic acid detection kit (PCR-fluorescent probe method) represented by Shanghai Bojie Medical Technology Co., Ltd., and the Salmonella and Shigella nucleic acid detection kit (fluorescent PCR method) represented by Jiangsu ShuoShi Biotechnology Co., Ltd. The above methods are all fluorescent PCR methods, which require specialized technicians to operate in a PCR laboratory, thus limiting their widespread use in primary hospitals.

[0009] Because existing products cannot simultaneously handle multiple pathogens, are simple to operate and require (suitable for primary care), and have high sensitivity and specificity, there is an urgent need for a product with significant comprehensive performance advantages that is easy to operate (fully automated, no human intervention), covers multiple pathogens with gastrointestinal infection-related symptoms, has high sensitivity, accuracy and specificity, and is cost-controllable, in order to meet market demands as medical institutions and needs become more widespread. Summary of the Invention

[0010] To address the shortcomings of existing products that cannot simultaneously detect multiple pathogens, require simple operation, and demand high sensitivity and specificity, this application provides a novel primer-probe composition for detecting gastrointestinal pathogens, a microfluidic chip technology platform including the primer-probe composition for fully automated detection, and a matching reagent kit composition. Using the primer-probe composition or reagent kit composition, multiplex detection of 32 gastrointestinal pathogens can be achieved, sufficiently covering syndrome-related pathogens, with high detection accuracy, sensitivity, and specificity. Furthermore, the entire detection process can be automated using fully automated equipment. With the appropriate equipment, the entire detection process can be automated, making it usable even by grassroots personnel. The combined application of multiple technologies ensures ease of operation, sensitivity, accuracy, and specificity. Moreover, the system has the potential for industrialization and is cost-effective.

[0011] To this end, the first aspect of this application provides a primer-probe composition for detecting gastrointestinal pathogens, the primer-probe composition comprising composition 1 and composition 2; composition 1 comprises a universal tag primer and 22 sets of primer-probe sets for detecting 15 gastrointestinal pathogens (including common viruses and bacteria) to be detected, and composition 2 comprises a universal tag primer and 17 sets of primer-probe sets for detecting 17 other gastrointestinal pathogens (bacteria and parasites) to be detected; wherein each set of primer-probe sets comprises an upstream primer, a downstream primer and a probe, and the 5' end of the upstream primer and the downstream primer is connected to the nucleotide sequence of the universal tag primer, the nucleotide sequence of the universal tag primer being shown in SEQ ID NO:121.

[0012] In some embodiments, the 5' end of the universal tag primer is modified with biotin.

[0013] For multiplex systems, controlling competition and interference between different primers and inhibiting primer dimer formation are key challenges. Previous studies have demonstrated that introducing universal primers can largely control competition and interference between different primers. A method for detecting 18 pathogens has been published in a related technology. This method uses a traditional multiplex system design without introducing universal primers. According to the final results, the sensitivity only reached 500 copies / reaction. Converted to the original sample, taking a 0.2 mL sample as an example, with a nucleic acid extraction and elution volume of 50 μL, even assuming a 100% nucleic acid extraction efficiency, the sensitivity based on the original sample would only reach 25,000 copies / mL, which is still relatively low.

[0014] For the design of universal primers, existing literature and patents mostly employ universal primer pairs. This involves adding a universal sequence to the 5' end of both the forward and reverse specific primers. Generally, the Tm value of the universal primers is higher than that of the specific primers. Two-stage amplification is then used to reduce competition and interference between multiple primers. This method still faces challenges in amplification efficiency, and primer dimers can still cause suboptimal amplification efficiency. Existing literature reports a method using a single tag to reduce primer dimers, and related patents also use a single universal primer (GTACGACTCACTATAGGGA), achieving simultaneous detection of 16 respiratory pathogens with a sensitivity of 1000 copies / mL, an order of magnitude higher than detection without universal primers. However, analysis of this universal primer reveals a risk of dimer formation, as shown below, which may affect the amplification efficiency of the system.

[0015]

[0016] Therefore, in response to the above situation, this application redesigned a universal tag primer with the nucleotide sequence ATACGACTCACTCTTGCGA (SEQ ID NO:121). This sequence does not bind to the genome sequences of human genomes, gastrointestinal pathogens within the detection range, or cross-pathogens tested and verified. Moreover, compared with previously reported sequences, this sequence is less likely to form dimers, as shown below, which is more conducive to the amplification of multiplex systems.

[0017]

[0018] The sequence was ligated to the 5' end of the designed forward and reverse specific primers to form labeled specific primers. The specific primers labeled with the tag were purified by PAGE, and the 5' end of the tag was labeled with biotin and purified by HPLC. Detection results showed that, compared with products from conventional low-repetition fluorescence PCR platforms, the detection performance using this universally tagged primer was better, despite covering a wider range of pathogens.

[0019] In some embodiments, the nucleotide sequences of the upstream primers, downstream primers, and probes in the 22 sets of primers and probes used to detect 15 gastrointestinal pathogens in composition 1 are shown in SEQ ID NO: 1 to 66, respectively; and the nucleotide sequences of the upstream primers, downstream primers, and probes in the 17 sets of primers and probes used to detect other 17 gastrointestinal pathogens in composition 2 are shown in SEQ ID NO: 67 to 117, respectively.

[0020] It is worth noting that the nucleotide sequences of the upstream and downstream primers and probes claimed in this application are not limited to the above-mentioned range. Shifting a few base positions or mismatching a few bases in the primers and probes is also within the scope of protection of this application.

[0021] In this application, the term "primer" refers to an oligonucleotide that is capable of "initiating" DNA synthesis by a template-dependent DNA polymerase, i.e., the 3'-terminus of the oligonucleotide provides a free 3'-OH group, which can be linked to more "nucleotides" by the template-dependent DNA polymerase to establish a 3' to 5' phosphodiester bond, thereby using deoxynucleoside triphosphate and thereby releasing pyrophosphate.

[0022] In this application, the term "upstream primer" refers to an oligonucleotide that extends continuously along the negative strand; the term "downstream primer" refers to an oligonucleotide that extends continuously along the positive strand. It should be understood that when the designations of the positive and negative strands are interchanged, the corresponding names of the upstream and downstream primers can also be interchanged. That is, the upstream and downstream primers in this application are relative terms.

[0023] In this application, the upstream and downstream primers and probes in the primer-probe set are designed for conserved regions of each pathogen, ensuring inclusiveness and accuracy within the pathogen. Furthermore, through comparative analysis and optimized design, the specificity among different pathogens was confirmed, and the dual design of primers and probes ensures the specificity of target recognition. Further, the upstream and downstream primers in the primer-probe set of this application are modified with universally tagged primers. Utilizing the effect of the same tag reduces competitive inhibition between primers and the influence of primer dimers in the multiplex system, thereby improving amplification efficiency and detection sensitivity.

[0024] In some embodiments, the 5' end of the probe is modified with an amino group (NH2).

[0025] In some embodiments, both composition 1 and composition 2 further include a set of primer-probe sets targeting the internal reference gene, which includes an upstream primer targeting the internal reference gene, a downstream primer targeting the internal reference gene, and a probe targeting the internal reference gene.

[0026] In this application, by setting an internal reference gene, the sampling process and the entire detection process can be monitored to ensure the accuracy of the detection process. In this application, the internal reference gene selected is RnaseP.

[0027] In some embodiments, the nucleotide sequences of the upstream primer, downstream primer, and probe targeting the internal reference gene are shown in SEQ ID NO:118-120, respectively.

[0028] In some embodiments, the 5' end of the probe targeting the internal reference gene is modified with an amino group (NH2).

[0029] It should be noted that in this application, only the universal tag primers are modified with biotin, while the 40 pairs of upstream and downstream primers targeting the internal reference gene and the gastrointestinal pathogens to be detected do not require modification with biotin, which effectively reduces the detection cost.

[0030] In this application, the 15 gastrointestinal pathogens to be detected are norovirus GI, norovirus GII, rotavirus (groups A / B / C, untyped), enteric adenovirus (types 40 / 41, untyped), astrovirus, zarovirus (types I / II / IV / V, untyped), Salmonella, Shigella, enterotoxigenic Staphylococcus aureus, enteroaggregative Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), and enterohemorrhagic Escherichia coli. Escherichia coli (EHEC), enteric invasive Escherichia coli (EIEC), and Escherichia coli O157:H7; the other 17 gastrointestinal pathogens to be detected are Campylobacter jejuni, Campylobacter coli, Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Listeria monocytogenes, Bacillus cereus, Clostridium difficile (A / B), Yersinia enterocolitica, Aeromonas hydrophila, Cronobacter sakazakii, Clostridium perfringens, Proteus mirabilis, Cryptosporidium, Entamoeba histolytica, Giardia lamblia, and Cyclospora.

[0031] In this application, the above-mentioned enteric aggregation Escherichia coli (EAEC), enteric pathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), and enteric invasive Escherichia coli (EIEC) are five diarrhea-causing Escherichia coli species.

[0032] The primer and probe composition described in this application is capable of detecting the above-mentioned 32 gastrointestinal pathogens, including norovirus GI, norovirus GII, rotavirus (groups A / B / C, untyped), enteric adenovirus (types 40 / 41, untyped), astrovirus, zarovirus (types I / II / IV / V, untyped), Salmonella, Shigella, enterotoxigenic Staphylococcus aureus, Escherichia coli O157:H7, Campylobacter jejuni, Campylobacter coli, Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Listeria monocytogenes, Bacillus cereus, and Clostridium difficile. A set of specific primer-probe pairs was developed for each of the following species: (A / B), Yersinia enterocolitica, Aeromonas hydrophila, Cronobacter sakazakii, Clostridium perfringens, Proteus mirabilis, Cryptosporidium, Entamoeba histolytica, Giardia lamblia, and Cyclospora. A total of 12 specific primer-probe pairs were developed for the detection of E. coli, including E. coli aggregates (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), and invasive E. coli (EIEC). The 12 Escherichia coli species were identified as *E. coli* elt, *E. coli* stp, *E. coli* sth, *E. coli* Eae, *E. coli* bfpB, *E. coli* Stx1, *E. coli* Stx2, *E. coli* coliipaH, *E. coli* uidA, *E. coli* aggR, *E. coli* astA, and *E. coli* pic. The presence or absence of five diarrhea-causing Escherichia coli species was determined by detecting these 12 species.

[0033] The primer and probe composition described in this application covers common viruses, bacteria, and parasites that cause gastrointestinal infection symptoms, providing a broad range of coverage and a more reliable basis for the effective implementation of clinical treatment.

[0034] The second aspect of this application provides a kit composition for fully automated detection of gastrointestinal pathogens, comprising the primer and probe composition as described in the first aspect of this application.

[0035] In some embodiments, the kit composition includes a kit 1 for detecting the 15 gastrointestinal pathogens to be detected and a kit 2 for detecting the other 17 gastrointestinal pathogens to be detected; the kit 1 includes nucleic acid extraction reagent, amplification reagent 1 and hybridization chromogenic reagent 1, and the kit 2 includes nucleic acid extraction reagent, amplification reagent 2 and hybridization chromogenic reagent 2.

[0036] In this application, the nucleic acid extraction reagents in kit 1 and kit 2 have the same composition.

[0037] In this application, the nucleic acid extraction reagent is used to extract nucleic acids from the sample to be tested. Amplification reagent 1 contains all primers in composition 1, and amplification reagent 2 contains all primers in composition 2. These are used to perform multiplex PCR amplification of the nucleic acids in the sample to be tested, thereby obtaining amplification product 1 and amplification product 2. Hybridization chromogenic reagent 1 contains all probes in composition 1, and hybridization chromogenic reagent 2 contains all probes in composition 2. The probes are used to capture target sequences in the corresponding amplification products, thereby immobilizing the target sequences in the amplification products at specific sites on the hybridization membrane for subsequent chromogenic development.

[0038] In some embodiments, the kit 1 further includes a positive control 1 and a negative control 1; the kit 2 further includes a positive control 2 and a negative control 2.

[0039] This application utilizes the aforementioned positive and negative control materials to effectively control the test results, avoid false positives and false negatives during the testing process, and further ensure the accuracy of the test results.

[0040] In some embodiments, the nucleic acid extraction reagent includes a lysis buffer, a lysis aid, a nucleic acid precipitation aid, magnetic beads, a binding buffer, a washing buffer, and an elution buffer.

[0041] In this application, the nucleic acid extraction reagent is used to obtain target nucleic acids from a sample. Specifically, the lysis buffer is used to disrupt the structure of pathogens in the sample, thereby fully releasing the nucleic acids within the sample; the lysis aid and nucleic acid precipitation aid are used to assist the lysis buffer, improving lysis efficiency and nucleic acid yield; the magnetic beads are used to adsorb the released nucleic acids; the binding solution is used to promote the binding of the released nucleic acids to the magnetic beads; the washing solution is used to purify the nucleic acids, removing impurities (proteins, etc.); and the elution solution is used to separate the magnetic beads from the nucleic acids, thereby releasing the purified nucleic acids, which are then dissolved in a liquid phase (e.g., water).

[0042] In some embodiments, the lysis buffer includes a guanidine salt, a surfactant, and a first buffer salt; the lysis aid is proteinase K; the nucleic acid precipitation aid is carrier RNA; the magnetic beads are silanol magnetic beads; the binding solution is isopropanol; the rinsing solution includes a second buffer salt and an organic alcohol; and the elution solution is water (e.g., purified water).

[0043] In some specific embodiments, the guanidine salt is guanidine isothiocyanate, and the concentration of guanidine isothiocyanate in the lysis buffer is 2-8 M; the surfactant is Triton X-100, and the concentration of Triton X-100 in the lysis buffer is 1-10 wt%; the first buffer salt is MOPS (3-morpholinopropanesulfonic acid), and the concentration of MOPS in the lysis buffer is 20-100 mM.

[0044] In some preferred embodiments, the lysis buffer contains 5 M guanidine isothiocyanate, 4 wt% Triton X-100, and 100 mM MOPS.

[0045] In some specific embodiments, the second buffer salt is MOPS, and the concentration of MOPS in the rinsing solution is 5-20 mM; the organic alcohol is ethanol, and the volume concentration of ethanol is 40-70%.

[0046] In some preferred embodiments, the rinsing solution is a 70 v / v ethanol solution containing 10 mM MOPS.

[0047] This application utilizes the nucleic acid extraction reagents described above to effectively obtain the target nucleic acid from the sample to be tested, which can then be used for subsequent detection.

[0048] In some embodiments, the amplification reagent 1 includes reverse transcriptase, Taq enzyme, UNG enzyme, dNTPs, specific primer 1, and universal tag primers. The specific primer 1 consists of 23 upstream primers and 23 downstream primers from composition 1 targeting the internal reference gene and 15 gastrointestinal pathogens to be detected. The amplification reagent 2 includes Taq enzyme, UNG enzyme, dNTPs, specific primer 2, and universal tag primers. The specific primer 2 consists of 18 upstream primers and 18 downstream primers from composition 2 targeting the internal reference gene and 17 other gastrointestinal pathogens to be detected. In this application, the Taq enzyme, UNG enzyme, dNTPs, and universal tag primers in amplification reagent 1 and amplification reagent 2 are identical.

[0049] In this application, amplification reagent 1 and amplification reagent 2 are used to perform multiplex PCR amplification on the target nucleic acid obtained by lysis, thereby amplifying the corresponding target sequence in the target nucleic acid.

[0050] In some embodiments, the amplification reagent 1 contains 1–5 U of reverse transcriptase, 1–3 U of Taq enzyme, 0.03–1 U of UNG enzyme, and 0.2–0.3 mM of dNTPS. Each primer in specific primer 1 contains 0.005–5 μM independently, and the universal tag primer contains 0.1–5 μM. The amplification reagent 2 contains 1–3 U of Taq enzyme, 0.03–1 U of UNG enzyme, and 0.2–0.3 mM of dNTPS. Each primer in specific primer 2 contains 0.005–5 μM independently, and the universal tag primer contains 0.1–5 μM.

[0051] In this application, since the gastrointestinal pathogens detected by kit 1 contain RNA viruses, amplification reagent 1 contains reverse transcriptase, which is an RNA-dependent DNA polymerase that uses RNA as a template to guide the synthesis of complementary DNA (cDNA) from deoxyribonucleotide triphosphates. The Taq polymerase in amplification reagent 1 and amplification reagent 2 is a thermostable DNA polymerase; the UNG enzyme is a uracil-N-glycosylation enzyme, whose function is to selectively hydrolyze and break uracil glycosidic bonds in double-stranded or single-stranded DNA containing dU, forming DNA chains with missing bases. These DNA chains are further hydrolyzed and broken under alkaline media and high temperatures, thus being eliminated. To facilitate the reverse transcription reaction, the UNG enzyme selected in this system is a heat-sensitive UNG enzyme, which loses its activity at 50°C. By employing Taq polymerase and UNG enzyme, this application can ensure the accuracy of PCR results and prevent non-specific PCR amplification and contamination.

[0052] This application achieves the reduction of competitive interference and primer dimer formation in multiplex amplification systems primarily by decreasing the amount of specific primers and increasing the amount of universal tag primers. Therefore, in designing the multiplex system, the content of each primer in specific primer 1 of amplification reagent 1 is 0.005–5 μM, and the content of the universal tag primer is 0.1–5 μM. Similarly, the content of each primer in specific primer 2 of amplification reagent 2 is 0.005–5 μM, and the content of the universal tag primer is 0.1–5 μM. By controlling the content of the universal tag primer and the content of each specific primer in amplification reagents 1 and 2 within the above ranges, the competitive interference and primer dimer formation in the multiplex amplification system can be significantly reduced, thereby improving the amplification effect.

[0053] In some preferred embodiments, the content of each primer in the specific primer 1 of the amplification reagent 1 is 0.1 μM, and the content of the universal tag primer is 1 to 5 μM; the content of each primer in the specific primer 2 of the amplification reagent 2 is 0.1 μM, and the content of the universal tag primer is 1 to 5 μM.

[0054] In some further preferred embodiments, the content of each primer in the specific primer 1 of the amplification reagent 1 is 0.1 μM, and the content of the universal tag primer is 2 μM; the content of each primer in the specific primer 2 of the amplification reagent 2 is 0.1 μM, and the content of the universal tag primer is 2 μM.

[0055] By further controlling the content of the universal tag primers in amplification reagent 1 and amplification reagent 2 and the content of each primer in the specific primers within the above range, the amplification effect can be improved.

[0056] In some specific embodiments, the amplification reagent 1 contains 2 U of reverse transcriptase, 2 U of Taq enzyme, 0.1 U of UNG enzyme, and 0.3 mM of dNTPS; each primer in specific primer 1 contains 0.1 μM; and the universal tag primer contains 2 μM. The amplification reagent 2 contains 2 U of Taq enzyme, 0.1 U of UNG enzyme, and 0.3 mM of dNTPS; each primer in specific primer 2 contains 0.1 μM; and the universal tag primer contains 2 μM.

[0057] This application achieves optimal amplification results by controlling the content of each component in the amplification reagent within the aforementioned range.

[0058] In some embodiments, the hybridization chromogenic reagent 1 includes a hybridization membrane 1, a hybridization washing solution, an enzyme conjugate solution, and a chromogenic solution, wherein the hybridization membrane 1 is immobilized with 23 probes from the composition 1 targeting the internal reference gene and 15 gastrointestinal pathogens to be detected; the hybridization chromogenic reagent 2 includes a hybridization membrane 2, a hybridization washing solution, an enzyme conjugate solution, and a chromogenic solution, wherein the hybridization membrane 2 is immobilized with 18 probes from the composition 2 targeting the internal reference gene and 17 other gastrointestinal pathogens to be detected. In this application, the compositions of the washing solution, enzyme conjugate solution, and chromogenic solution in the hybridization chromogenic reagent 1 and the hybridization chromogenic reagent 2 are identical.

[0059] In this application, hybridization chromogenic reagent 1 and hybridization chromogenic reagent 2 are used to hybridize and chromogenically develop corresponding multiplex PCR amplification products. Specifically, a specific probe is immobilized on the hybridization membrane, which can specifically bind to the corresponding target sequence in the amplification product, thereby immobilizing the target sequence at a specific site on the hybridization membrane. The hybridization washing buffer is used to provide a hybridization environment and remove interference from non-specific substances in the amplification product. The enzyme in the enzyme conjugate solution can be immobilized on the target sequence through biotin-streptavidin binding. The chromogenic solution reacts with the enzyme in the enzyme conjugate solution, thereby chromogenically developing the target sequence.

[0060] In some embodiments, the hybridization membrane 2 and the content of each probe on the hybridization membrane 2 are each independently 0.2 to 50 μM, preferably 50 μM.

[0061] In this application, the probe can be spotted onto a hybridization membrane using nanoliter spotting technology. The corresponding multiplexed products amplified in a single tube are then hybridized with a corresponding hybridization membrane covering the pathogens being detected, enabling the simultaneous detection of multiple gastrointestinal pathogens in a single tube.

[0062] In some embodiments, both hybridization membrane 1 and hybridization membrane 2 are nylon membranes; the hybridization rinsing solution includes sodium dodecyl sulfate and a third buffer salt; the enzyme conjugation solution includes streptavidin-labeled horseradish peroxidase; and the colorimetric solution includes urea peroxide, a fourth buffer salt, and tetramethylbenzidine.

[0063] In some specific embodiments, the concentration of sodium dodecyl sulfate in the hybridization rinsing solution is 5–15 wt%; the third buffer salt is phosphate, and the concentration of phosphate in the hybridization rinsing solution is 10–100 mM; the concentration of streptavidin-labeled horseradish peroxidase (HRP) in the enzyme conjugate solution is (0.1–1) μg / mL; the concentration of urea oxychloride in the chromogenic solution is 0.01–0.1 wt%; the fourth buffer salt is citrate, and the concentration of citrate in the chromogenic solution is 5–50 mM; and the concentration of tetramethylbenzidine (TMB) in the chromogenic solution is 0.01–0.05 wt%.

[0064] In some preferred embodiments, the hybridization rinsing solution contains 10 wt% SDS and 10 mM phosphate; the enzyme conjugate solution contains 0.4 μg / mL streptavidin-labeled horseradish peroxidase; and the colorimetric solution contains 0.03 wt% urea peroxide, 5 mM citrate, and 0.015 wt% TMB.

[0065] In some embodiments, both hybridization membrane 1 and hybridization membrane 2 are further immobilized with chromogenic control probes (SP probes); preferably, the content of chromogenic control probes on hybridization membrane 1 and hybridization membrane 2 is 0.2–50 μM. In some specific embodiments, chromogenic control probes are immobilized at three positioning points in both hybridization membrane 1 and hybridization membrane 2.

[0066] The SP probe described in this application is used for quality control of hybridization colorimetric results to ensure the accuracy of the detection results. In addition to its colorimetric quality control function, the SP probe also serves as a positioning point for automatic identification and interpretation by the software. The design of three positioning points enables the transition from manual visual interpretation to automatic software identification. After the experiment, the automatic interpretation software searches for and locates the three fixed positioning points (SP points) to determine the location of other spots. A simulated circle is then defined based on the size of the SP points. The average grayscale value of the pixels within the simulated circle is used as the spot brightness information, and the average grayscale value of the four surrounding areas is used as the background brightness. The difference in grayscale between the spot brightness and the background brightness is the contrast value of that point. The deeper the color of the spot, the greater the grayscale difference between the spot and the surrounding area, and consequently, the higher the contrast value. Therefore, the contrast value reflects the spot detection result.

[0067] In some embodiments, the positive control 1 is a mixture of enteroaggregative Escherichia coli positive strains and norovirus GII pseudovirus; the positive control 2 is a mixture of Clostridium perfringens positive bacteria and Clostridium difficile positive bacteria; and both negative control 1 and negative control 2 are HEK293 cells.

[0068] In this application, the positive control 1 was positive for norovirus GII, and positive for E. coli aggR, E. coli astA and E. coli pic sites; the positive control 2 was positive for Clostridium perfringens and Clostridium difficile; the negative control 1 and negative control 2 were negative for all pathogens in the combination.

[0069] In this application, the principle of using kit 1 and kit 2 in the kit composition to detect the corresponding gastrointestinal pathogens belongs to the nucleic acid extraction-amplification-reverse dot blot hybridization method. Specifically, the target nucleic acid is obtained from the sample using a nucleic acid extraction reagent; the target sequence is amplified using an amplification reagent; and the amplified product is bound to the probe of the hybridization membrane through hybridization. Since the primers are modified with biotin, the product bound to the hybridization membrane also carries biotin modification. Through the interaction of biotin and avidin, the amplified product can bind to HRP modified with streptavidin. Then, the colorimetric reaction of hydrogen peroxide and TMB catalyzed by HRP is used to achieve visual detection results. If the sample contains a target, the product enriched and amplified after extraction and amplification will bind to the hybridization membrane containing the specific probe, and then, through the colorimetric effect of HRP-TMB, a blue spot will appear at the probe position.

[0070] The kit composition described in this application immobilizes specific probes for various gastrointestinal pathogens and internal standard probes on corresponding hybridization membranes using nanometer-scale dot membrane technology. Therefore, the kit composition described in this application can simultaneously detect 32 gastrointestinal pathogens, and the presence of the corresponding gastrointestinal pathogen in the sample can be determined based on the color development of each probe position on the hybridization membrane. Simultaneously, the kit composition combines... Figure 3-4 The equipment shown, such as Figure 5-6 When used with the chip shown, the entire detection process can be automated, avoiding operational errors or contamination introduced by manual intervention. It has good repeatability and high stability, and is suitable for screening and testing in grassroots institutions.

[0071] The third aspect of this application provides a method for detecting gastrointestinal pathogens using a kit composition as described in the second aspect of this application.

[0072] In some embodiments, the method includes the following steps:

[0073] S1, The target nucleic acid in the sample to be tested is obtained using the nucleic acid extraction reagent;

[0074] S2, the target nucleic acid is mixed with amplification reagent 1 and amplification reagent 2 respectively to obtain amplification system 1 and amplification system 2; then multiplex PCR amplification is performed on amplification system 1 and amplification system 2 respectively to obtain amplification product 1 and amplification product 2.

[0075] S3, hybridize and develop color on the amplification product 1 using the hybridization chromogenic reagent 1, hybridize and develop color on the amplification product 2 using the hybridization chromogenic reagent 2, and interpret the color development results.

[0076] In this application, the target nucleic acid in the sample to be tested in step S1 is obtained by magnetic bead method. After the sample is lysed by lysis buffer, the released nucleic acid is captured by magnetic beads. After washing and elution, the purified target nucleic acid is obtained.

[0077] In some embodiments, in step S2, both the multiplex PCR amplification of the amplification system 1 and the multiplex PCR amplification of the amplification system 2 are performed in a single tube.

[0078] In this application, the conditions for multiplex PCR amplification of the amplification system 1 are different from those for multiplex PCR amplification of the amplification system 1.

[0079] In some specific embodiments, the conditions for multiplex PCR amplification of the amplification system 1 are as follows:

[0080] 35℃ for 3 minutes;

[0081] 50℃ for 10 minutes;

[0082] 95℃ for 30 seconds;

[0083] 95℃ for 15 seconds, 55℃ for 20 seconds, 72℃ for 15 seconds, 45 cycles;

[0084] 72℃ for 1 minute.

[0085] In some embodiments, the conditions for multiplex PCR amplification of the amplification system 2 are as follows:

[0086] 35℃ for 3 minutes;

[0087] 95℃ for 30 seconds;

[0088] 95℃ for 15 seconds, 60℃ for 20 seconds, 72℃ for 15 seconds, 45 cycles;

[0089] 72℃ for 1 minute.

[0090] In some specific embodiments, step S3 is specifically performed as follows: hybridize the amplification product 1 with hybridization membrane 1 at 30℃~50℃ for 10~25 min, and hybridize the amplification product 2 with hybridization membrane 2 at the same temperature for 30℃~50℃ for 10~25 min; then add hybridization washing solution to wash hybridization membrane 1 and hybridization membrane 2 respectively, and then add enzyme conjugation solution to react at 30~37℃ for 10~15 min, add color development solution (urea peroxide + TMB) to react for 5~10 min to develop color, and interpret the color development results.

[0091] In some implementations, the method is performed automatically by fully automated equipment.

[0092] The method described in this application can be completed automatically by fully automated equipment, achieving a completely unmanned process. This avoids operational errors or contamination introduced by human intervention, and offers good repeatability and high stability, making it suitable for screening and testing in grassroots institutions. In some specific embodiments, the fully automated equipment can use the applicant's microfluidic nucleic acid chip detection equipment.

[0093] Specifically, the characteristics of the detection method described in this application compared with other methods are shown in Table 1.

[0094] Table 1

[0095]

[0096]

[0097] As shown in Table 1, compared with existing products, this application has significant advantages in terms of pathogen coverage and automation. Compared with domestic multi-test products, it has a clear advantage in pathogen coverage, and the automation function avoids the possibility of contamination from manual operation. Compared with leading international multi-test companies, it not only has more comprehensive pathogen coverage but also has an advantage in cost control.

[0098] This application includes at least one of the following beneficial technical effects:

[0099] (1) Sensitivity: The sensitivity of the optimized single-tube multiplex detection system in this application is consistent with or slightly higher than that of conventional real-time fluorescent PCR. This indicates that the method in this application can cover a wider range of pathogens in a single detection, and there is no significant loss in sensitivity due to the implementation of multiplex detection. This can maximize the freedom of operators and benefit patients.

[0100] (2) Accuracy: The method described in this application was compared with conventional real-time fluorescent PCR. The results showed that the positive and negative concordance rates of the single-tube multiplex detection method in this application and the conventional real-time fluorescent PCR detection results were both 100%. This indicates that the method described in this application can cover a wider range of pathogens in a single detection and has high accuracy.

[0101] (3) Specificity: The kits 1 and 2 in the kit composition of this application were used to detect pathogens outside the detection target range, and the results were negative, which further confirmed that the kits described in this application have high specificity.

[0102] (4) Ease of Operation: During the testing process, the only operation involved in this application is adding the sample to the sample pool corresponding to the detection chip; all other operations are completed by the equipment, thereby reducing contamination caused by human intervention and further improving accuracy. Compared with non-automated products involving a large number of operations, this product has better user-friendliness. Attached Figure Description

[0103] Figure 1 This is a dot matrix distribution diagram on hybridization membrane 1 of the kit composition described in Example 2, showing the detection probes and chromogenic control probes for the 15 gastrointestinal pathogens and internal reference genes targeted by kit 1; the meanings of the symbols in the diagram are as follows:

[0104] SP: Colorimetric control probe; IC: Detection probe targeting the internal reference gene; the others are detection probes targeting 15 gastrointestinal pathogens, among which 13 detection targets (E. coli) were set for 5 diarrheal Escherichia coli (including O157:H7), and the combination was used to determine the 5 diarrheal Escherichia coli (including O157:H7).

[0105] Figure 2 This is a dot matrix distribution diagram on hybridization membrane 2 of the detection probes and chromogenic control probes for the 17 gastrointestinal pathogens and internal reference genes targeted by kit 2 in the kit composition described in Example 2; the meanings of the symbols in the figure are as follows:

[0106] SP: Colorimetric control probe; IC: Detection probe targeting the internal reference gene; others are detection probes targeting 17 gastrointestinal pathogens.

[0107] Figure 3 This is an external view of the automated equipment used in Example 3.

[0108] Figure 4 This is an internal structure diagram of the automated equipment used in Example 3.

[0109] Figure 5 This is a front view of the chip installed in the device described in Example 3.

[0110] Figure 6 This is a reverse view of the chip installed in the device described in Example 3.

[0111] Figure 7 This is an electrophoresis result of the amplification products from Example 1, which were amplified under the same conditions using three different primers.

[0112] Figure 8 This is a diagram showing the design of the concentration combination of specific primers and tags in Example 2.

[0113] Figure 9 This is an electrophoresis result of the amplified product in Example 2. Detailed Implementation

[0114] To make this application easier to understand, the following detailed description is provided in conjunction with embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods. Unless otherwise indicated, the nucleic acid sequences in this application are written from left to right in a 5' to 3' direction.

[0115] Example 1: Design of primer-probe composition

[0116] Download the following strains from the NCBI database: Norovirus GI, Norovirus GII, Rotavirus (Groups A / B / C), Enteric Adenovirus (Types 40 / 41), Astrovirus, Zarovirus (Types I / II / IV / V), Salmonella, Shigella, Enterotoxigenic Staphylococcus aureus, Enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), Enterohemorrhagic Escherichia coli (EHEC), and Invasive Escherichia coli. The genome sequences of *Escherichia coli* (EIEC), *Escherichia coli* O157:H7, *Campylobacter jejuni*, *Campylobacter coli*, *Vibrio cholerae*, *Vibrio vulnificus*, *Vibrio parahaemolyticus*, *Listeria monocytogenes*, *Bacillus cereus*, *Clostridium difficile* (A / B), *Yersinia enterocolitica*, *Aeromonas hydrophila*, *Cronobacter sakazakii*, *Clostridium perfringens*, *Proteus mirabilis*, *Cryptosporidium*, *Cyclospora*, *Entamoeba hemorrhagicum*, and *Giardia lamblia* were sequenced and aligned. Primers and probes were then designed based on the conserved gene regions of these pathogens. After preliminary design, primer interactions were analyzed to remove designs with five or more bases matching at their 3' ends. Similarly, probe interactions with the amplification products of each target site were analyzed to remove designs with multiple consecutive bases matching.

[0117] The primers and probes designed above were then analyzed against the pathogens intended for cross-reactivity in the study to eliminate the possibility of false positives at the design level. The pathogens with cross-reactivity verified in this application include: varicella-zoster virus, Epstein-Barr virus, Escherichia coli, Stenotrophomonas maltophilia, Burkholderia cepacia, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus salivarius, Acinetobacter baumannii, Klebsiella pneumoniae, Cryptococcus, Serratia marcescens, Citrobacter, Aspergillus fumigatus, and Aspergillus flavus. Comparative analysis showed that this design did not exhibit cross-reactivity with the aforementioned pathogens.

[0118] To control competition and interference between different primers in the multiplex system and to inhibit primer dimer formation, a novel universal tag primer (Tag) with the nucleotide sequence ATACGACTCACTCTTGCGA (SEQ ID NO: 121) was designed in this application. This sequence does not bind to the genomes of human pathogens, gastrointestinal pathogens within the detection range, or the genomes of pathogens with verified cross-reactivity. Furthermore, compared to previously reported sequences, this sequence is less prone to dimer formation, thus being more conducive to amplification in the multiplex system. Linking this sequence to the 5' end of the designed forward and reverse specific primers to form labeled specific primers significantly reduces competition and interference between different primers in the multiplex system and improves amplification efficiency. The Tag-labeled specific primers were purified by PAGE, and the 5' end of the Tag was labeled with biotin and purified by HPLC.

[0119] To further verify the improvement in amplification efficiency brought about by the universal tag primer designed in this application, the amplification efficiency of primers without tags, primers labeled with existing single universal primers (GTACGACTCACTATAGGGA), and primers labeled with the universal tag primer designed in this application were compared under the same conditions. The results are as follows: Figure 7 As shown. Figure 7 In the diagram, A represents the electrophoresis band of the amplification product labeled with the universally tagged primers of this application; B represents the electrophoresis band of the amplification product labeled with existing single universal primers; and C represents the electrophoresis band of the amplification product labeled with untagged primers. Figure 7 The electrophoresis results of the three groups of amplification products show that, compared with conventional primers without tag labels, both groups of tag-labeled primers (A and B) can improve amplification efficiency, and the tag provided in this application is even better at improving amplification efficiency.

[0120] In this application, RnaseP is selected as the internal reference gene (internal standard) for detection, used to monitor the sampling status and the entire detection process.

[0121] After theoretical analysis and experimental verification, the nucleotide sequences of the primers and probes finally selected in this application are shown in Tables 2 and 3.

[0122] Table 2: Nucleotide sequences of primers and probes in composition 1 of the primer-probe composition described in this application.

[0123]

[0124]

[0125]

[0126] Table 3: Nucleotide sequences of primers and probes in composition 2 of the primer-probe composition described in this application.

[0127]

[0128]

[0129] Example 2: Design of a Multiple Detection System

[0130] I. Design of the multiplex detection system of kit 1 in the kit composition

[0131] 1. Conditions for nucleic acid extraction

[0132] Nucleic acid extraction is achieved using the magnetic bead method. After the sample is lysed with lysis buffer, the released nucleic acid is captured by magnetic beads. After washing and elution, purified nucleic acid is obtained.

[0133] In this embodiment, the preferred nucleic acid extraction conditions are as follows: 0.2 mL of sample is added, and lysed with 0.2 mL of lysis buffer containing 4% Triton X-100, 5 M guanidine isothiocyanate, 100 mM MOPS, 0.2 mg proteinase K, and 6 μg carrier RNA at 56 °C for 10 min; then 0.2 mL of isopropanol and 0.2 mg of silanol magnetic beads are added, and the mixture is allowed to bind for 10 min; the magnetic beads are then washed with 70 v / v ethanol containing 10 mM MOPS; then purified water is added, and the nucleic acid is eluted by heating at 56 °C for 5 min.

[0134] 2. Amplification System 1

[0135] In this application, the reduction of competitive interference and primer dimer formation in the multiplex amplification system is achieved primarily by reducing the amount of specific primers and increasing the amount of tag used. Therefore, in designing the multiplex amplification system 1, the content of each specific primer is 0.005-0.5 μM, and the content of the tag is 0.1-5 μM.

[0136] The concentrations of specific primers and tags were optimized through combination design, such as... Figure 8 As shown. Multiplex amplification was performed using each concentration group, along with singlex amplification. The electrophoresis results of the amplification products are shown below. Figure 9 As shown. Comparing the electrophoresis results of the amplification products of each group, and comparing them with the effect of singleton amplification, it can be confirmed that groups 7-9 basically meet the requirements. Among them, group 8 has the highest cost performance. Therefore, the content of each specific primer in amplification system 1 is preferably 0.1 μM, and the content of Tag is preferably 1-5 μM (most preferably 2 μM).

[0137] After optimization, the content of each specific primer in amplification system 1 was 0.1 μM, the content of Tag was 2 μM, the content of reverse transcriptase was 2 U, the content of Taq enzyme was 2 U, the content of dNTPS was 0.3 mM, and the content of UNG enzyme was 0.1 U.

[0138] Using the primers in composition 1 designed in Example 1, amplification system 1 was prepared according to the content of each component of the optimized amplification system 1. The extracted nucleic acid was used for single-tube multiplex PCR amplification to obtain amplification product 1.

[0139] The amplification conditions were as follows: 35℃ for 3 min; 50℃ for 10 min; 95℃ for 30 s; (95℃ for 15 s, 55℃ for 20 s, 72℃ for 15 s), 45 cycles; 72℃ for 1 min.

[0140] 3. Hybridization system

[0141] In this embodiment, probes for the 15 pathogens detected by kit 1 are spotted onto the hybridization membrane using nanometer spotting technology. The dot matrix distribution of each pathogen, as well as the detection probes for the internal reference gene and the chromogenic quality control probes on hybridization membrane 1, is shown in the figure below. Figure 1 As shown in Table 4, the rules for determining each target point on hybridization membrane 1 were written into the interpretation software. The rules for positive results are shown in Table 4. After the experiment, the interpretation software provided the interpretation results based on the color development or combination of spots at each location on hybridization membrane 1.

[0142] Table 4: Positive interpretation rules for hybridization membrane 1 in kit 1

[0143]

[0144]

[0145] Note 1: (+) indicates that a hybridization spot was detected at the probe location, and (-) indicates that a hybridization spot was not detected at the probe location.

[0146] Note 2: Supplementary explanation for the interpretation of positive results for 5 types of diarrhea-causing Escherichia coli:

[0147] (1) Given that virulence genes can be transferred between bacteria, when astA, pic and other virulence genes are positive at the same time, astA and pic are not used as the basis for type determination. For example, if uidA, ipaH, astA and pic are positive at the same time, it is determined to be EIEC. If uidA, elt, astA and pic are positive at the same time, it is determined to be ETEC.

[0148] (2) O157:H7 is a typical strain of EHEC. The O157:H7 detection site is positive and can be directly identified as O157:H7.

[0149] (3) There is an overlap between EIEC and Shigella. Shigella has virulence / non-virulence types, which is reflected in whether it contains a virulence plasmid. The virulence plasmid contains the invasion-related ipaH gene. When ipaH and Shigella are both positive, Shigella is the primary interpreter, i.e., the virulence of Shigella.

[0150] (4) When the result is determined to be positive for EHEC, if Eae is positive, it means that the sample contains typical EHEC; if Eae is negative, it means that the sample contains atypical EHEC.

[0151] (5) When the result is determined to be positive for EPEC, if bfpB is positive, it means that the sample contains typical EPEC; if bfpB is negative, it means that the sample contains atypical EPEC.

[0152] (6) When Salmonella (-), Shigella (-), all five diarrhea-causing Escherichia coli (-), and uidA (+), it indicates that the sample to be tested is positive for Escherichia coli of common type.

[0153] Multiple products amplified by a single tube are hybridized with a hybridization membrane 1 covering the pathogens to be detected, enabling the simultaneous detection of multiple pathogens in a single tube.

[0154] In this embodiment, the preferred hybridization system is as follows: the concentration of each target detection probe on hybridization membrane 1 is 50 μM; the concentration of SDS in the hybridization washing solution is 10 wt%, and the buffer salt is phosphate with a concentration of 10 mM; the concentration of HPR in the enzyme conjugate solution is 0.4 μg / mL; the concentration of urea peroxide in the colorimetric solution is 0.03 wt%; the buffer salt is citrate with a concentration of 5 mM; the concentration of TMB is 0.015%, and the buffer salt is citrate with a concentration of 5 mM.

[0155] After amplification, amplification product 1 was hybridized with hybridization membrane 1 at 50℃ for 25 min. Hybridization washing buffer was added to wash hybridization membrane 1, followed by the addition of enzyme conjugation solution. The mixture was reacted at 37℃ for 10 min, and then chromogenic solution (urea peroxide + TMB) was added, reacting for 5 min. Depending on the template added, chromogenic spots could be observed at different locations on hybridization membrane 1.

[0156] II. Design of the multiplex detection system for reagent kit 2 in the reagent kit composition

[0157] 1. Conditions for nucleic acid extraction

[0158] Nucleic acid extraction is achieved using the magnetic bead method. After the sample is lysed with lysis buffer, the released nucleic acid is captured by magnetic beads. After washing and elution, purified nucleic acid is obtained.

[0159] In this embodiment, the nucleic acid extraction conditions of kit 2 are exactly the same as those of kit 1. Therefore, the target nucleic acid obtained by a single nucleic acid extraction can be used for multiplex amplification in both kit 1 and kit 2.

[0160] 2. Amplification System 2

[0161] In this application, the reduction of competitive interference and primer dimer formation in the multiplex amplification system is achieved primarily by reducing the amount of specific primers and increasing the amount of tag used. Therefore, in designing the multiplex amplification system 2, the content of each specific primer is 0.005-0.5 μM, and the content of the tag is 0.1-5 μM.

[0162] Based on the optimization results of amplification system 1, the preferred concentration of each specific primer in amplification system 2 is 0.1 μM, and the preferred concentration of Tag is 1-5 μM (most preferably 2 μM). Therefore, in the optimized amplification system 2, the concentration of each specific primer is 0.1 μM, the concentration of Tag is 2 μM, the concentration of Taq enzyme is 2 U, the concentration of dNTPS is 0.3 mM, and the concentration of UNG enzyme is 0.1 U.

[0163] Using the primers in combination 2 designed in Example 1, amplification system 2 was prepared according to the content of each component of the optimized amplification system 2. The extracted nucleic acid was used for single-tube multiplex PCR amplification to obtain amplification product 2.

[0164] The amplification conditions were as follows: 35℃ for 3 min; 95℃ for 30 s; (95℃ for 15 s, 60℃ for 20 s, 72℃ for 15 s), 45 cycles; 72℃ for 1 min.

[0165] 3. Hybrid System 2

[0166] In this embodiment, probes for the 17 pathogens detected by kit 2 are spotted onto the hybridization membrane using nanometer spotting technology. The dot matrix distribution of each pathogen, as well as the detection probes for the internal reference gene and the chromogenic quality control probes on hybridization membrane 2, is shown in the figure below. Figure 2 As shown in Table 5, the rules for determining each target point on hybridization membrane 2 were written into the interpretation software. The rules for positive results are shown in Table 5. After the experiment, the interpretation software provided the interpretation results based on the color development or combination of spots at each location on hybridization membrane 2.

[0167] Table 5: Positive interpretation rules for hybridization membrane 2 in kit 2

[0168]

[0169]

[0170] Note: (+) indicates that a hybridization spot was detected at the probe location, and (-) indicates that a hybridization spot was not detected at the probe location.

[0171] Multiple products amplified by a single tube are hybridized with a hybridization membrane 2 covering the pathogens to be detected, enabling the simultaneous detection of multiple pathogens in a single tube.

[0172] In this embodiment, the preferred hybridization system is as follows: the concentration of each target detection probe on the hybridization membrane 2 is 50 μM; the concentration of SDS in the hybridization washing solution is 10 wt%, and the buffer salt is phosphate with a concentration of 10 mM; the concentration of HPR in the enzyme conjugate solution is 0.4 μg / mL; the concentration of urea peroxide in the colorimetric solution is 0.03 wt%; the buffer salt is citrate with a concentration of 5 mM; the concentration of TMB is 0.015%, and the buffer salt is citrate with a concentration of 5 mM.

[0173] After amplification, amplification product 2 was hybridized with hybridization membrane 2 at 50℃ for 25 min. Hybridization washing buffer was added to wash hybridization membrane 2, followed by the addition of enzyme conjugation solution. The mixture was reacted at 37℃ for 10 min, and then chromogenic solution (urea peroxide + TMB) was added, reacting for 5 min. Depending on the template added, chromogenic spots could be observed at different locations on hybridization membrane 2.

[0174] Example 3: Detection of 32 Gastrointestinal Pathogens

[0175] Simulated samples of 15 gastrointestinal pathogens to be detected were prepared using artificially synthesized pseudoviruses and inactivated pathogen cultures from Kit 1. These simulated samples were diluted to 1000 copies / mL. Nucleic acid extraction, amplification, and hybridization were performed on the simulated samples according to the multiplex detection system conditions of Kit 1 in Example 2. The detection results are shown in Table 6. Simulated samples of 17 gastrointestinal pathogens to be detected were prepared using inactivated pathogen cultures or artificially synthesized plasmids from Kit 2. These simulated samples were diluted to 1000 copies / mL. Nucleic acid extraction, amplification, and hybridization were performed on the simulated samples according to the multiplex detection system conditions of Kit 2 in Example 2. The detection results are shown in Table 7.

[0176] The above-mentioned testing process was carried out automatically using automated equipment. The automated equipment used was a nucleic acid chip detector manufactured by Beijing Bohui Innovation Biotechnology Group Co., Ltd., and its appearance and internal structure diagrams are shown below. Figure 2 and 3 As shown; front and back views of the chip installed inside the device are shown below. Figure 4 and 5 As shown. The chip used in this embodiment has been granted patents (CN201110235199.2, CN201110235234.0, CN201210121023.9, CN201220175387.0).

[0177] Table 6: Detection of various pathogen targets by kit 1

[0178]

[0179] As shown in Table 6, Kit 1 can detect all target pathogens normally, that is, this system can achieve a sensitivity level of 1000 copies / mL.

[0180] Table 7: Detection of various pathogen targets by kit 2

[0181]

[0182]

[0183] As shown in Table 7, Kit 2 can detect all target pathogens normally, that is, this system can achieve a sensitivity level of 1000 copies / mL.

[0184] Example 4: Performance Evaluation

[0185] The automated equipment used in this embodiment is the same as that in Embodiment 3.

[0186] 1. Performance evaluation of reagent kit 1

[0187] (1) Accuracy testing

[0188] For the remaining clinical samples (40 cases), parallel comparison detection was performed using real-time fluorescence PCR (qPCR) and the method described in this application (using the multiplex detection system of kit 1 determined in Example 2 combined with automated equipment). The results are shown in Table 8.

[0189] Table 8: Comparison of the detection method of kit 1 described in this application with that of real-time fluorescent PCR

[0190]

[0191]

[0192]

[0193]

[0194] As shown in Table 8, the positive and negative concordance rates of the single-tube multiplex detection of the kit 1 in this application and the conventional real-time fluorescence PCR detection results are both 100%, indicating that the detection method using the kit 1 in this application has high accuracy.

[0195] (2) Sensitivity detection

[0196] Nine of the remaining 40 clinical samples were serially diluted and then compared in parallel using real-time fluorescence PCR (qPCR) and the method described in this application (using the multiplex detection system of kit 1 determined in Example 2 combined with automated equipment). The results are shown in Table 9.

[0197] Table 9: Comparison of the detection method of kit 1 described in this application with that of real-time fluorescent PCR

[0198]

[0199]

[0200] As shown in Table 9, the sensitivity of the single-tube multiplex detection in Kit 1 of this application is consistent with, or slightly higher than, that of conventional real-time fluorescent PCR. This means that using the method described in this application not only allows for a wider range of pathogens to be covered in a single detection, but also does not result in a significant loss of sensitivity due to multiplexing, thus maximizing the convenience for operators and benefiting patients.

[0201] (3) Specific detection

[0202] Using the multiplex detection system of kit 1 determined in Example 2, combined with automated equipment, the cross-interference effect of pathogens outside the detection target range of kit 1 was tested. The results are shown in Table 10. The pathogens outside the detection target range include 13 pathogen-positive strains (Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Campylobacter jejuni, Campylobacter coli, Clostridium perfringens, Yersinia enterocolitica, Clostridium difficile toxin, Bacillus cereus, Listeria monocytogenes, Aeromonas hydrophila, Cronobacter sakazakii, Proteus mirabilis) and 10 other common intestinal pathogen-positive strains (Alcaligenes faecalis, Enterococcus faecalis, Streptococcus pyogenes, Lactobacillus acidophilus, Bifidobacterium longum, Proteus vulgaris, Citrobacter freundii, Pseudomonas aeruginosa, Enterobacter cloacae, Serratia marcescens).

[0203] Table 10: Specificity Detection Results

[0204]

[0205]

[0206] As shown in Table 10, the multiplex detection system of Kit 1 yielded negative results for pathogens outside the detection target range. This indicates that pathogens outside the detection target range did not interfere with the detection results, and the multiplex detection system of Kit 1 in this application possesses good specificity.

[0207] 2. Performance evaluation of reagent kit 2

[0208] (1) Accuracy testing

[0209] For the remaining clinical samples (40 cases), parallel comparison detection was performed using real-time fluorescence PCR (qPCR) or first-generation sequencing (since some pathogens detected in kit 2 do not have commercially available products for comparison (such as Proteus mirabilis), first-generation sequencing was used for comparison) and the method described in this application (using the multiplex detection system of kit 2 determined in Example 2 combined with automated equipment). The results are shown in Table 11.

[0210] Table 11: Comparison of the detection method of kit 2 described in this application with that of real-time fluorescent PCR

[0211]

[0212]

[0213]

[0214] As shown in Table 11, the positive and negative concordance rates of the single-tube multiplex detection of the kit 2 in this application are both 100% with those of conventional real-time fluorescence PCR or first-generation sequencing, indicating that the detection method using the kit 2 in this application has high accuracy.

[0215] (2) Sensitivity detection

[0216] Five of the remaining 40 clinical samples were serially diluted and then compared in parallel using real-time fluorescence PCR (qPCR) and the method described in this application (using the multiplex detection system of kit 2 determined in Example 2 combined with automated equipment). The results are shown in Table 12.

[0217] Table 12: Comparison of detection methods of kit 2 described in this application with real-time fluorescent PCR results

[0218]

[0219] As shown in Table 12, the sensitivity of the single-tube multiplex detection in Kit 2 of this application is consistent with, or slightly higher than, that of conventional real-time fluorescence PCR. This means that using the method described in this application not only allows for a wider range of pathogens to be covered in a single detection, but also does not result in a significant loss of sensitivity due to multiplexing, thus maximizing the convenience for operators and benefiting patients.

[0220] (3) Specific detection

[0221] Using the multiplex detection system of kit 2 determined in Example 2, combined with automated equipment, the cross-interference effect of pathogens outside the detection target range of kit 2 was tested. The results are shown in Table 13. The pathogens outside the detection target range included 15 pathogens (Norovirus GI / GII, Rotavirus, Enteric Adenovirus, Astrovirus, Zarovirus, Salmonella, Shigella, Staphylococcus aureus, Enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), Enterohemorrhagic Escherichia coli (EHEC), Invasive Escherichia coli (EIEC), O157:H7) and 10 other common enteric pathogen positive strains (Alcaligenes faecalis, Enterococcus faecalis, Streptococcus pyogenes, Lactobacillus acidophilus, Bifidobacterium longum, Proteus vulgaris, Citrobacter freundii, Pseudomonas aeruginosa, Enterobacter cloacae, Serratia marcescens).

[0222] Table 13: Specific Detection Results

[0223]

[0224]

[0225] As shown in Table 13, the multiplex detection system of Kit 2 yielded negative results for pathogens outside the detection target range. This indicates that pathogens outside the detection target range did not interfere with the detection results, and the multiplex detection system of Kit 2 in this application possesses good specificity.

[0226] In summary, this application achieves comprehensive pathogen coverage and fully automated detection without sacrificing performance, and outperforms conventional platform products in terms of sensitivity, accuracy, specificity, and interference tolerance.

[0227] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. A primer probe composition for gastrointestinal pathogen detection, characterized by, The primer probe composition comprises composition 1 and composition 2; the composition 1 comprises one universal tag primer and 22 groups of primer probe groups respectively used for detecting 15 gastrointestinal pathogens to be detected; the composition 2 comprises one universal tag primer and 17 groups of primer probe groups respectively used for detecting other 17 gastrointestinal pathogens to be detected; each group of the primer probe group comprises an upstream primer, a downstream primer and a probe, and the 5' end of the upstream primer and the downstream primer is connected with the nucleotide sequence of the universal tag primer, and the nucleotide sequence of the universal tag primer is shown as SEQ ID NO: 121; the nucleotide sequences of the upstream primer, the downstream primer and the probe in the 22 groups of primer probe groups respectively used for detecting 15 gastrointestinal pathogens to be detected in the composition 1 are shown as SEQ ID NO: 1~66; the nucleotide sequences of the upstream primer, the downstream primer and the probe in the 17 groups of primer probe groups respectively used for detecting other 17 gastrointestinal pathogens to be detected in the composition 2 are shown as SEQ ID NO: 67~117.

2. The primer probe composition of claim 1, wherein, The 5' end of the universal tag primer is modified with biotin.

3. The primer probe composition of claim 1, wherein, The 5' end of the probe is modified with amino.

4. The primer probe composition according to claim 1 or 2, characterized in that, The composition 1 and the composition 2 further each comprise one group of primer probe group for the internal reference gene, which comprises one upstream primer for the internal reference gene, one downstream primer for the internal reference gene and one probe for the internal reference gene.

5. The primer probe composition of claim 4, wherein, The nucleotide sequences of the upstream primer, the downstream primer and the probe for the internal reference gene are shown as SEQ ID NO: 118~120; the 5' end of the probe for the internal reference gene is modified with amino.

6. The primer probe composition according to claim 1 or 2, characterized in that, The 15 gastrointestinal pathogens to be detected are Norovirus GI, Norovirus GII, Rotavirus, Enteric Adenovirus, Astrovirus, Sapovirus, Salmonella, Shigella, Staphylococcus aureus enterotoxigenic, Enteropathogenic Escherichia coli, Enteropathogenic Escherichia coli, Enterotoxigenic Escherichia coli, Enterohemorrhagic Escherichia coli, Enteroinvasive Escherichia coli and Escherichia coli O157:H7; the other 17 gastrointestinal pathogens to be detected are Campylobacter jejuni, Campylobacter coli, Vibrio cholera, Vibrio vulnificus, Vibrio parahaemolyticus, Listeria monocytogenes, Bacillus cereus, Clostridium difficile, Yersinia enterocolitica, Aeromonas hydrophila, Enterobacter sakazakii, Clostridium perfringens, Proteus mirabilis, Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis.

7. A kit for automatic detection of gastrointestinal pathogens, comprising the primer probe composition according to any one of claims 1-6.

8. The kit of claim 7, wherein The kit comprises kit 1 for detecting 15 gastrointestinal pathogens to be detected and kit 2 for detecting other 17 gastrointestinal pathogens to be detected; the kit 1 comprises nucleic acid extraction reagent, amplification reagent 1 and hybridization color developing reagent 1, and the kit 2 comprises nucleic acid extraction reagent, amplification reagent 2 and hybridization color developing reagent 2.

9. The kit of claim 8, wherein The kit 1 further comprises positive control 1 and negative control 1, and the kit 2 further comprises positive control 2 and negative control 2.

10. The kit of claim 8, wherein The nucleic acid extraction reagent comprises lysis solution, lysis aid, nucleic acid aid, magnetic beads, binding solution, rinsing solution and elution solution.

11. The kit of claim 10, wherein The lysis solution comprises guanidine salt, surfactant and first buffer salt; the lysis aid is proteinase K; the nucleic acid aid is Carrier RNA; the magnetic beads are silicon hydroxyl magnetic beads; the binding solution is isopropanol; the rinsing solution comprises second buffer salt and organic alcohol; and the elution solution is water.

12. The kit of claim 11, wherein The guanidine salt is guanidine isothiocyanate, and the concentration of the guanidine isothiocyanate in the lysis solution is 2-8 M; the surfactant is Triton X-100, and the concentration of the Triton X-100 in the lysis solution is 1-10 wt%; and the first buffer salt is MOPS, and the concentration of the MOPS in the lysis solution is 20-100 mM.

13. The kit of claim 11, wherein The second buffer salt is MOPS, and the concentration of the MOPS in the rinsing solution is 5-20 mM; and the organic alcohol is ethanol, and the volume concentration of the ethanol is 40-70%.

14. The kit of claim 8, wherein The amplification reagent 1 comprises reverse transcriptase, Taq enzyme, UNG enzyme, dNTPs, specific primer 1 and universal tag primer, and the specific primer 1 is 23 upstream primers and 23 downstream primers in the composition 1 for the internal reference gene and the 15 gastrointestinal pathogens to be detected; and the amplification reagent 2 comprises Taq enzyme, UNG enzyme, dNTPs, specific primer 2 and universal tag primer, and the specific primer 2 is 18 upstream primers and 18 downstream primers in the composition 2 for the internal reference gene and the other 17 gastrointestinal pathogens to be detected.

15. The kit of claim 14, wherein The content of the reverse transcriptase in the amplification reagent 1 is 1-5 U, the content of the Taq enzyme is 1-3 U, the content of the UNG enzyme is 0.03-1 U, the content of the dNTPS is 0.2-0.3 mM, the content of each primer in the specific primer 1 is independently 0.005-5 μM, and the content of the universal tag primer is 0.1-5 μM; and the content of the Taq enzyme in the amplification reagent 2 is 1-3 U, the content of the UNG enzyme is 0.03-1 U, the content of the dNTPS is 0.2-0.3 mM, the content of each primer in the specific primer 2 is independently 0.005-5 μM, and the content of the universal tag primer is 0.1-5 μM.

16. The kit of claim 15, wherein The content of each primer in the specific primer 1 in the amplification reagent 1 is 0.1 μM, and the content of the universal tag primer is 1-5 μM. The content of each primer in the specific primer 2 in the amplification reagent 2 is 0.1 μM, and the content of the universal tag primer is 1-5 μM.

17. The kit of claim 8, wherein The hybridization color developing reagent 1 comprises a hybridization membrane 1, a hybridization rinsing solution, an enzyme binding solution and a color developing solution, wherein the hybridization membrane 1 is fixed with 23 probes for the internal reference gene and the 15 gastrointestinal pathogens to be detected in the composition 1; the hybridization color developing reagent 2 comprises a hybridization membrane 2, a hybridization rinsing solution, an enzyme binding solution and a color developing solution, wherein the hybridization membrane 2 is fixed with 18 probes for the internal reference gene and the other 17 gastrointestinal pathogens to be detected in the composition 2.

18. The kit of claim 17, wherein The content of each probe on the hybridization membrane 1 and the hybridization membrane 2 is independently 0.2-50 μM.

19. The kit of claim 17, wherein The hybridization membrane 1 and the hybridization membrane 2 are nylon membranes; the hybridization rinsing solution comprises sodium dodecyl sulfate and a third buffer salt; the enzyme binding solution comprises streptavidin-labeled horseradish peroxidase; and the color developing solution comprises urea peroxide, a fourth buffer salt and tetramethylbenzidine.

20. The kit of claim 19, wherein The concentration of the sodium dodecyl sulfate in the hybridization rinsing solution is 5-15 wt%; the third buffer salt is phosphate, and the concentration of the phosphate in the hybridization rinsing solution is 10-100 mM; the concentration of the streptavidin-labeled horseradish peroxidase in the enzyme binding solution is (0.1-1) μg / mL; the concentration of urea peroxide in the color developing solution is 0.01-0.1 wt%; the fourth buffer salt is citrate, and the concentration of the citrate in the color developing solution is 5-50 mM; and the concentration of tetramethylbenzidine in the color developing solution is 0.01-0.05 wt%.

21. The kit of claim 17, wherein The hybridization membrane 1 and the hybridization membrane 2 are further fixed with color developing quality control probes.

22. The kit of claim 21, wherein The content of the color developing quality control probes on the hybridization membrane 1 and the hybridization membrane 2 is 0.2-50 μM.

23. The kit of claim 9, wherein The positive quality control 1 is a mixture of enteric aggregative Escherichia coli positive strain and norovirus GII pseudovirus; the positive quality control 2 is a mixture of Clostridium perfringens positive strain and Clostridium difficile positive strain; and the negative quality control 1 and the negative quality control 2 are both HEK293 cells.

24. A method for detecting gastrointestinal pathogens for non-disease diagnosis purposes by using the kit according to any one of claims 8-23.

25. The method of claim 24, wherein, The method comprises the following steps: S1, obtaining target nucleic acids in a sample to be detected by using the nucleic acid extraction reagent; S2, mixing the target nucleic acids with the amplification reagent 1 and the amplification reagent 2 respectively to obtain amplification systems 1 and 2; then performing multiplex PCR amplification on the amplification systems 1 and 2 respectively to obtain amplification products 1 and 2; S3, performing hybridization color development on the amplification product 1 by using the hybridization color developing reagent 1, performing hybridization color development on the amplification product 2 by using the hybridization color developing reagent 2, and interpreting the color development results.

26. The method according to claim 25, wherein the multiplex PCR amplification on the amplification system 1 and the multiplex PCR amplification on the amplification system 2 in step S2 are both completed in a single tube.

27. The method of any of claims 24-26, wherein, The method is automatically completed by a fully automatic device. The method is automatically completed by a fully automatic device.

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