Infectious endocarditis pathogen detection kit based on digital PCR platform, application and detection method

Abstract

The invention discloses an infectious endocarditis pathogen detection kit based on a digital PCR (Polymerase Chain Reaction) platform, application and a detection method. The kit comprises a primer probe mixed solution, a PCR premixed solution, a bacterial solution containing an internal standard sequence fragment, and a positive quality control product and a negative quality control product of inactivated engineering bacteria containing a target gene fragment. The kit is used for detecting 15 infectious endocarditis pathogenic microorganisms and methicillin drug-resistant genes mecA and mecC in one tube. In practical application tests, even if the concentration of pathogens is as low as less than 10 copies per milliliter, the pathogens can be accurately identified, and extremely high sensitivity is shown. And synchronous detection of multiple pathogens and drug resistance thereof can be rapidly completed within 2 hours without waiting for a blood culture result.

Description

Technical Field

[0001] The present invention relates to a kit and method for detecting pathogens of infective endocarditis, and in particular to a kit and method for detecting pathogens of infective endocarditis based on a digital PCR platform. Background Art

[0002] Infective endocarditis (IE) is an inflammation of the endocardium, valves, or adjacent aorta caused by pathogens such as bacteria and fungi, accompanied by systemic pathology. IE can be categorized as acute or subacute. Subacute IE is more common clinically, often caused by low-virulence pathogens, with a gradual onset and a prolonged course. However, acute endocarditis is more acute and has a shorter course. The pathogens rapidly proliferate and spread within the heart, rapidly damaging it. Without active treatment, death often occurs within weeks. In recent years, the incidence of endocarditis has significantly increased with the aging population, the increase in the number of elderly patients with degenerative valvular heart disease, and the increased use of prosthetic heart valve replacement, device implantation, and various intravascular procedures. Despite advances in treatment and management, the in-hospital mortality rate remains high (20-40%), necessitating rapid early diagnosis.

[0003] The diagnosis of this disease is based on the Duke criteria, which include clinical manifestations, laboratory results, and echocardiography. The main basis of these criteria is the detection of vegetations on the heart valves by echocardiography and positive blood cultures. However, echocardiography is positive in only 50-94% of cases, while blood cultures are negative in 2.5-31% of IE cases, resulting in undiagnosed infection. Reasons for negative blood cultures may include previous cardiac antibiotic treatment, slow-growing or unculturable microorganisms, fungi, and / or intracellular microorganisms. It is very important to identify the pathogen that causes endocarditis because this helps guide antibiotic treatment for the disease. In addition to the above methods, metagenomics next-generation sequencing (mNGS) has also been studied for the detection of pathogenic microorganisms in patients with IE. Its sensitivity is higher than that of blood culture, but its clinical use has certain limitations. Compared with conventional pathogen detection products, products based on mNGS technology currently have the following characteristics: a more complex detection process, susceptibility to interference from human genes, longer detection time, high professional requirements for result interpretation, and high detection costs. It is recommended that clinicians first use traditional microbiological tests and conventional molecular biology products to detect common pathogens through tentative diagnosis, and not blindly use mNGS technology. However, conventional PCR detection products have very single targets and uneven compatible instruments, resulting in a narrow target indication for this type of product. These products are only applicable when the screening purpose is extremely strong and the target is clear. In addition, due to the low sensitivity of qPCR, the number of pathogenic microorganisms in the blood is not high, which can easily lead to missed detection. If pathogens can be identified as early as possible and appropriate anti-infection programs can be initiated, the patient's prognosis can be improved, especially for those with severe infections. Pathogen detection is of great significance in infection determination. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a detection kit and method for infective endocarditis pathogens based on a digital PCR platform.

[0005] The causative microorganisms of infective endocarditis are primarily bacteria, but fungi, viruses, and chlamydia can also be involved. The pathogens vary depending on the site of infection, the source of bacteremia, and host risk factors. The oral cavity, skin, and lower respiratory tract are relatively common targets for viridans streptococci, staphylococci, and HACEK organisms (Haemophilus species, Actinomyces, Cardiomyces, Eikenella, and Kingella). Hospital-acquired endocarditis is primarily caused by Staphylococcus aureus, coagulase-negative staphylococci, and fastidious Gram-negative bacilli. Intravenous endocarditis is primarily caused by Staphylococcus aureus. Among these, the majority of staphylococci that cause endocarditis are methicillin-resistant. Overall, 80% to 90% of cases are caused by streptococci or Staphylococcus aureus, with the remainder primarily caused by enterococci and HACEK organisms (Haemophilus species, Actinomyces, Cardiomyces, Eikenella, and Kingella).

[0006] Technical solution: On the one hand, the present invention provides an infective endocarditis pathogen detection kit based on a digital PCR platform, which comprises a primer-probe mixture, a PCR premix, a bacterial solution containing an internal standard sequence fragment, a positive quality control product of an inactivated engineered bacteria containing a target gene fragment, and a negative quality control product.

[0007] The components of the kit for detecting the pathogen of infective endocarditis are shown in the table below: Component name Ingredients Primer probe mixture Combinations of primers and probes provided in the examples PCR master mix <![CDATA[A solution containing deoxynucleoside triphosphate (dN(U)TP), MgCl2, DNA polymerase (taq enzyme), and UDG]]> Internal standard Bacterial solution containing internal standard gene fragment Positive quality control Inactivated engineered bacteria containing target gene fragments Negative control water According to the NCBI database, we searched for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, Enterococcus faecium, Enterococcus faecalis, Haemophilus influenzae, Streptococcus mitis, Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus gallolyticus, Streptococcus pyogenes, and Streptococcus agalactiae. agalactiae) gene sequence, and a gene sequence with good specificity, high intraspecific conservation and appropriate fragment length was selected as the target sequence for detection through blast comparison; Staphylococcus aureus sequence: AGAGCGTGCTGGATTTGTATCAAAAACAGATGATTATTTTTATAACTTTATTGACACATATGGAGATAAAGTATTAGTACCATTAGCATATATTGACCTTGATGAATATGTGTTAAAGTTGCAACAGGAATTGAATGACAAAGAAAATCGTCGTGATCAAATGATGGC Staphylococcus epidermidis sequences: ATATTACGTAGGTCAAGTTCAATCTTCTGACTTACCACAACAAAGCTCATCTGATACGCTACCGAATACTGGTATGAAAGATAAAAATTTCAATACAAATGGTATAATAAGTCTATTTTTACTCACTGCTGGTTTCATTACACTTTACC Staphylococcus haemolyticus sequence: AAACAAACTATGGAAATCCATCATGACAAACATCACAACACTTATGTTACCAAATTAAATTCTGCAGTTGAGGGAACAGATCTTGAATCTAAATCAATTGAAGAAATTGTTGCTAACTTAGATAGTGTACCTGAAGATAT Staphylococcus hominis sequence TAGATGGATCTGAAACAGTAGTATCAGGTGCTTCATGTACTACAAACTCATTAGCACCAGTTGCTAAAGTTTTAAATGACGAATTTGGTATTGTTGAAGG Staphylococcus capitis sequence: TCAGATATTCAAACTGCAGTACGTAATAATGGTGGCGGTCACTTAAACCACTCATTATTCTGGGAATTATTATCACCAAATTCTGAAGAAAAAGGTGAAGTAG Enterococcus faecium sequence: AGAGCGTGCTGGATTTGTATCAAAAACAGATGATTATTTTTATAACTTTATTGACACATATGGAGATAAAGTATTAGTACCATTAGCATATATTGACCTTGATGAATATGTGTTAAAGTTGCAACAGGAATTGAATGACAAAGAAAATCGTCGTGATCAAATGATGGC Enterococcus faecalis sequence: GAAAGATGTCGCTTTCTATGATTATGATGCAAAATACATCAATAACACGATTGAAATGCAAATCCCAGCGCATGTTCCAGAAGAAGTAGCTCATCAAGCGCAAGAATACGCTAAAAAAGCGTATATTATGTTAGATGGAAGTGGCTTAAGTCGC Haemophilus influenzae sequences: AATGCGTGATGCTGGTTATGACGATGATATTTGCGGAGGAATTACTGCTGCTTCTTGTATTATTGGGCCATTAGTTCCACCGAGTATTGCAATGATTATTTACGGTGT(C / A)ATCCCAATGAATCTATCGCAAAACTCTT Streptococcus mitis sequence: GGAAATTACACGTGTAGGAGCAGATATCAAAATCAAATGTAGCAATTGTGACCATGTTGTCATGATGGGACGATATGATTTTGATCGAAAAATGAATAAAATTATTGACTGAAAACCCTTAGTTAGA Streptococcus mutans sequence: AGAGGAATATACCAATGTTGTGATTGCTAAGAATGTGGATAAGTTTGCGGAATGGGGTGTCACAGATTTTGAAATGGCACCGCAGTATGTGTCTTCAACAGATGGTTCTT Streptococcus sanguis sequence GACTGATGAGAAGATTACCAAGTGGTCAGCTAAACACTTCAACGGTACCAATATCTTAGGTCGTGGTGCTTACTATGTTCTTAAAGACTGGGCTAGCAACGAATATCTCAACA Streptococcus salivarius sequence: GGGAAGCATTATGGATTACCCTTATGCAACATGCTGTGGCCTTAATGACGAAGTGGCACATGCTTTTCCACGTCACTATATTTTGAAAGATGGTGACCTCTTGAAAGTTGATATGGTCTTGTCAGAACCTATCGATAAGTCAGTCCTTGATGTGT Gallolytic Streptococcus: AAAGCTGGTGAGGACCAAGCACTCAACCAAAAACGTGATAAATTGCTTAATCATAAAAACATTGCAGATACGTTGACCAATGCTTAGTCATGCTTGATGACGAAGAATTTTCAAGTCTATCTAACATTCGTTCAGCCATGAATGATTTGATGACGCTC Streptococcus pyogenes sequence: CTTATGTCGCTAATACCAATGCGGCTTTAGAAAAACACCCAGAGATCGGAGAAAATCTGGAGGAACTATTGGCAGATGTCACTAAGATTCCAGAAGATATT Streptococcus agalactiae sequence: GCAAAAGAACAGATGGAACAAAGTGGTTCAAAGTTCTTAGGTATTATTCTTAATAAAGTTAATGAATCTGTTGCTACTTACGGCGATTATGGAAATTACGGAAAAAGGTATAGAAAAAGGAAGTAAGGGG The gene sequences of drug-resistant genes mecA and mecC were retrieved from the NCBI database, and a gene sequence with good specificity, high conservation and appropriate fragment length was selected as the target sequence for detection through blast comparison; Methicillin-resistant gene mecA sequence: GTAGAAATGACTGAACGTCCGATAAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTT Methicillin-resistant gene mecC sequence: ATAGATGCTAGAGTACAAGAAAGTATTTATAAACATATGAAAAATGACGATGGATCTGGTACAGCATTACAACCAAAAACTG Internal standard sequence: TTGCTTGCGGGTGAGAGTGGTATTGAAGTAGTTGCAGTAAACGACTTAACAGACGACGATGTGCCGGGCTCCTGCATAAATATGACACTATGCAAGGGTCCTATCAGCTTGTTGGCG According to the target sequence and primer probe design principles, the designed primer probe sequences for each pathogen are as follows (probes are fluorescently labeled): Staphylococcus aureus F1 primer: 5'- AGA GCG TGC TGG ATT TGT AT -3' sequence 1 R1 primer: 5'- GCC ATC ATT TGA TCA CGA CG -3' sequence 2 P1 probe: 5'- GTC ATT CAA TTC CTG TTG CAA C -3' (MGB) sequence 3 Staphylococcus epidermidis F2 primer: CATATTACGTAGGTCAAGTTC sequence 4 R2 primer: GGTAAAGTGTAATGAAACCAG sequence 5 P2 probe: GCTCATCTGATACGCTACCGAATAC sequence 6 hemolytic Staphylococci F3 primer: AAACAAACTATGGAAATCCATC sequence 7 R3 primer: ATATCTTCAGGTACACTATCTAAG sequence 8 P3 probe: TTCTGCAGTTGAGGGAACAGATCT sequence 9 Staphylococcus hominis F4 primer: TAGATGGATCTGAAACAGTAGTAT sequence 10 R4 primer: CCTTCAACAATACCAAATTCGTC sequence 11 P4 probe: AGGTGCTTCATGTACTACAAACTCATTG sequence 12 Staphylococcus capitis F5 primer: TCAGATATTCAAACTGCAGTACG sequence 13 R5 primer: CTACTTCACCTTTTTCTTCAGA sequence 14 P5 probe: ATGGTGGCGGTCACTTAAACCA sequence 15 Enterococcus faecium F6 primer: TAGAGACATTGAATATGCC sequence 16 R6 primer: TCGAATGTGCTACAATC sequence 17 P6 probe: 5'-CGTATTGACCAGTGCATGT-3' (MGB) sequence 18 Enterococcus faecalis F7 primer: GAA AGA TGT CGC TTT CTA TGA TTA TGA sequence 19 R7 primer: GCG ACT TAA GCC ACT TCC ATC TA sequence 20 P7 probe: FAM-ATG CAA ATC CCA GCG CAT GTT CC-BHQ sequence 21 Haemophilus influenzae F8 primer: 5′-AATGCGTGATGCTGGTTATGAC sequence 22 R8 primer: 5′-AAGAGTTTTGCGATAGATTCATTGG sequence 23 P8 probe: TET-AGAAGCAGCAGTAATT-MGBNFQ sequence 24 Streptococcus mitis F9 primer: GGAAATTACACGTGTAGGA sequence 25 R9 primer: TCTAACTAAGGGTTTTCAGTCA sequence 26 P9 probe: CCATGTTGTCATGATGGGACGATA sequence 27 Streptococcus mutans F10 primer: AGAGGAATATACCAATGTTGTG sequence 28 R10 primer: AAGAACCATCTGTTGAAGAC sequence 29 P10 probe: TGTGGATAAGTTTGCGGAATGGG sequence 30 Streptococcus sanguis F11 primer: GACTGATGAGAAGATTACCA sequence 31 R11 primer: TGTTGAGATATTCGTTGCTA sequence 32 P11 probe: GGTCAGCTAAACACTTCAACGGTACC sequence 33 Streptococcus salivarius F12 primer: GGGAAGCATTATGGATTACC sequence 34 R12 primer: ACACATCAAGGACTGACTTATC sequence 35 P12 probe: ATGCAACATGCTGTGGCCTTAATGA 36 Streptococcus gallolyticus F13 primer: AAAGCTGGTGAGGACCAAGC sequence 37 R13 primer: GAGCGTCATCAAATCATTCA sequence 38 P13 probe: GCAGATACGTTGACCAATGCTTATG sequence 39 Streptococcus pyogenes F14 primer: AATATCTTCTGGAATCTTAGTGA sequence 40 R14 primer: CTTATGTCGCTAATACCAATG sequence 41 P14 probe: CACCCAGAGATCGGAGAAAATCTGGA sequence 42 Streptococcus agalactiae F15 primer: GCAAAAGAACAGATGGAACAAAGTG sequence 43 R15 primer: CCCCTTACTTCCTTTTTCTATACC sequence 44 P15 probe: CTGTTGCTACTTACGGCGATTATGG sequence 45 MecA F16 primer: GTAGAAATGACTGAACGTCCGATAA sequence 46 R16 primer: AACTTCCACATACCATCTTCT sequence 47 P16 probe: ACGTTGCGATCAATGTTACCGTAGT sequence 48 MecC F17 primer: ATAGATGCTAGAGTACAAGA sequence 49 R17 primer: TTTTGGTTGTAATGCTGTA sequence 50 P17 probe: CCAGATCCATCGTCATTTTTC sequence 51.

[0008] Internal standard F18 primer: TTGCTTGCGGGTGAGAGT sequence 52 R18 primer: CGCCAACAAGCTGATAGGAC sequence 53 P18 probe: ATGTGCCGGGCTCCTGCAT sequence 54.

[0009] The volume ratio of the primer-probe mixture and the PCR premix is 2-5:2-5, preferably 5:5; the volume ratio of the bacterial solution containing the internal standard sequence fragment, the positive quality control product of the inactivated engineered bacteria containing the target gene fragment, and the negative quality control product is: 10-50:10-300:10-300, preferably 20:50:50.

[0010] Compared with the prior art, the present invention has the following beneficial effects: (1) Dynamic monitoring of pathogen load Compared with traditional PCR products and gene chips, the method of the present invention can detect multiple pathogens in a single reaction. Traditional PCR products can only label one target per fluorescent channel, and the number of targets detected in a single-well reaction is limited by the number of fluorescent channels. Our reaction uses different fluorescent dyes to label probes for different targets, combined with primer probes of varying concentrations. After amplification, the individual targets are distinguished based on the combination of fluorescent signals within each microreaction unit. This kit can detect 15 pathogenic microorganisms of infective endocarditis and the methicillin-resistance genes mecA and mecC in a single tube. The pathogens include Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, Enterococcus faecium, Enterococcus faecalis, Haemophilus influenzae, Streptococcus mitis, Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus gallolyticus, Streptococcus pyogenes, and Streptococcus agalactiae. Multiple detections can also dynamically monitor the development of pathogens at a lower cost, providing a direct basis for clinical medication.

[0011] (2) Ultra-high sensitivity Digital PCR technology is theoretically capable of detecting a single copy of a pathogen. In practical testing, our solution has accurately identified pathogens even at concentrations as low as fewer than 10 copies per milliliter, demonstrating exceptionally high sensitivity and effectively reducing the risk of false negatives associated with other technologies. Digital PCR is a nucleic acid quantification technique used to precisely measure the absolute quantity of DNA or RNA. Unlike traditional PCR products, digital PCR directly counts target molecules by dividing the reaction system into thousands or even more independent, small-volume reaction units. This approach offers greater sensitivity and accuracy.

[0012] (3) Pathogen and drug resistance information Traditional drug resistance analysis requires waiting for blood culture before drug sensitivity testing, which is time-consuming. Our method not only detects pathogens but also adds the detection of drug resistance genes. With one tube and multiple targets, there is no need to culture bacteria first and then test the bacterial resistance like traditional blood culture. It can quickly complete the simultaneous detection of multiple pathogens and their drug resistance within 2 hours without waiting for blood culture results. This allows us to quickly obtain the drug resistance information of pathogens while detecting them, greatly shortening the diagnosis time and providing key support for timely and effective clinical treatment decisions. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the rendering of plane 1; Figure 2 This is the rendering of plane 2; Figure 3 This is the rendering of plane 3. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.

[0015] Determination of target fragments, primers, and probes According to the NCBI database, we searched for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis, Enterococcus faecium, Enterococcus faecalis, Haemophilus influenzae, Streptococcus mitis, Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivarius, Streptococcus gallolyticus, Streptococcus pyogenes, and Streptococcus agalactiae. agalactiae) gene sequence, and a gene sequence with good specificity, high intraspecific conservation and appropriate fragment length was selected as the target sequence for detection through blast comparison; Staphylococcus aureus sequence: AGAGCGTGCTGGATTTGTATCAAAAACAGATGATTATTTTTATAACTTTATTGACACATATGGAGATAAAGTATTAGTACCATTAGCATATATTGACCTTGATGAATATGTGTTAAAGTTGCAACAGGAATTGAATGACAAAGAAAATCGTCGTGATCAAATGATGGC Staphylococcus epidermidis sequences: ATATTACGTAGGTCAAGTTCAATCTTCTGACTTACCACAACAAAGCTCATCTGATACGCTACCGAATACTGGTATGAAAGATAAAAATTTCAATACAAATGGTATAATAAGTCTATTTTTACTCACTGCTGGTTTCATTACACTTTACC Staphylococcus haemolyticus sequence: AAACAAACTATGGAAATCCATCATGACAAACATCACAACACTTATGTTACCAAATTAAATTCTGCAGTTGAGGGAACAGATCTTGAATCTAAATCAATTGAAGAAATTGTTGCTAACTTAGATAGTGTACCTGAAGATAT Staphylococcus aureus sequences TAGATGGATCTGAAACAGTAGTATCAGGTGCTTCATGTACTACAAACTCATTAGCACCAGTTGCTAAAGTTTTAAATGACGAATTTGGTATTGTTGAAGG Staphylococcus capitis sequence: TCAGATATTCAAACTGCAGTACGTAATAATGGTGGCGGTCACTTAAACCACTCATTATTCTGGGAATTATTATCACCAAATTCTGAAGAAAAAGGTGAAGTAG Enterococcus faecium sequence: AGAGCGTGCTGGATTTGTATCAAAAACAGATGATTATTTTTATAACTTTATTGACACATATGGAGATAAAGTATTAGTACCATTAGCATATATTGACCTTGATGAATATGTGTTAAAGTTGCAACAGGAATTGAATGACAAAGAAAATCGTCGTGATCAAATGATGGC Enterococcus faecalis sequence: GAAAGATGTCGCTTTCTATGATTATGATGCAAAATACATCAATAACACGATTGAAATGCAAATCCCAGCGCATGTTCCAGAAGAAGTAGCTCATCAAGCGCAAGAATACGCTAAAAAAGCGTATATTATGTTAGATGGAAGTGGCTTAAGTCGC Haemophilus influenzae sequence: AATGCGTGATGCTGGTTATGACGATGATATTTGCGGAGGAATTACTGCTGCTTCTTGTATTATTGGGCCATTAGTTCCACCGAGTATTGCAATGATTATTTACGGTGT(C / A)ATCGCCAATGAATCTATCGCAAAACTCTT Streptococcus mitis sequence: GGAAATTACACGTGTAGGAGCAGATATCAAAATCAAATGTAGCAATTGTGACCATGTTGTCATGATGGGACGATATGATTTTGATCGAAAAATGAATAAAATTATTGACTGAAAACCCTTAGTTAGA Streptococcus mutans sequence: AGAGGAATATACCAATGTTGTGATTGCTAAGAATGTGGATAAGTTTGCGGAATGGGGTGTCACAGATTTTGAAATGGCACCGCAGTATGTGTCTTCAACAGATGGTTCTT Streptococcus sanguinis sequence: GACTGATGAGAAGATTACCAAGTGGTCAGCTAAACACTTCAACGGTACCAATATCTTAGGTCGTGGTGCTTACTATGTTCTTAAAGACTGGGCTAGCAACGAATATCTCAACA Streptococcus salivarius sequence: GGGAAGCATTATGGATTACCCTTATGCAACATGCTGTGGCCTTAATGACGAAGTGGCACATGCTTTTCCACGTCACTATATTTTGAAAGATGGTGACCTCTTGAAAGTTGATATGGTCTTGTCAGAACCTATCGATAAGTCAGTCCTTGATGTGT Gallolytic Streptococcus: AAAGCTGGTGAGGACCAAGCACTCAACCAAAAACGTGATAAATTGCTTAATCATAAAAACATTGCAGATACGTTGACCAATGCTTAGTCATGCTTGATGACGAAGAATTTTCAAGTCTATCTAACATTCGTTCAGCCATGAATGATTTGATGACGCTC Streptococcus pyogenes sequence: CTTATGTCGCTAATACCAATGCGGCTTTAGAAAAACACCCAGAGATCGGAGAAAATCTGGAGGAACTATTGGCAGATGTCACTAAGATTCCAGAAGATATT Streptococcus agalactiae sequence: GCAAAAGAACAGATGGAACAAAGTGGTTCAAAGTTCTTAGGTATTATTCTTAATAAAGTTAATGAATCTGTTGCTACTTACGGCGATTATGGAAATTACGGAAAAAGGTATAGAAAAAGGAAGTAAGGGG The gene sequences of drug-resistant genes mecA and mecC were retrieved from the NCBI database, and a gene sequence with good specificity, high conservation and appropriate fragment length was selected as the target sequence for detection through blast comparison; Methicillin-resistant gene mecA sequence: GTAGAAATGACTGAACGTCCGATAAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTT Methicillin-resistant gene mecC sequence: ATAGATGCTAGAGTACAAGAAAGTATTTATAAACATATGAAAAATGACGATGGATCTGGTACAGCATTACAACCAAAAACTG Internal standard sequence: TTGCTTGCGGGTGAGAGTGGTATTGAAGTAGTTGCAGTAAACGACTTAACAGACGACGATGTGCCGGGCTCCTGCATAAATATGACACTATGCAAGGGTCCTATCAGCTTGTTGGCG According to the target sequence and primer probe design principles, the designed primer probe sequences for each pathogen are as follows (probes are fluorescently labeled): Staphylococcus aureus F1 primer: 5'- AGA GCG TGC TGG ATT TGT AT -3' R1 primer: 5'- GCC ATC ATT TGA TCA CGA CG -3' P1 probe: 5'-GTC ATT CAA TTC CTG TTG CAA C -3' (MGB) Staphylococcus epidermidis F2 primer: CATATTACGTAGGTCAAGTTC R2 primer: GGTAAAGTGTAATGAAACCAG P2 probe: GCTCATCTGATACGCTACCGAATAC hemolytic Staphylococci F3 primer: AAACAAACTATGGAAATCCATC R3 primer: ATATCTTCAGGTACACTATCTAAG P3 probe: TTCTGCAGTTGAGGGAACAGATCT Staphylococcus hominis F4 primer: TAGATGGATCTGAAACAGTAGTAT R4 primer: CCTTCAACAATACCAAATTCGTC P4 probe: AGGTGCTTCATGTACTACAAACTCATTG Staphylococcus capitis F5 primer: TCAGATATTCAAACTGCAGTACG R5 primer: CTACTTCACCTTTTTCTTCAGA P5 probe: ATGGTGGCGGTCACTTAAACCA Enterococcus faecium F6 primer: TAGAGACATTGAATATGCC R6 primer: TCGAATGTGCTACAATC P6 probe: 5'-CGTATTGACCAGTGCATGT-3' (MGB) Enterococcus faecalis F7 primer: GAA AGA TGT CGC TTT CTA TGA TTA TGA R7 primer: GCG ACT TAA GCC ACT TCC ATC TA P7 probe: FAM-ATG CAA ATC CCA GCG CAT GTT CC-BHQ Haemophilus influenzae F8 primer: 5′-AATGCGTGATGCTGGTTATGAC R8 primer: 5′-AAGAGTTTTGCGATAGATTCATTGG P8 probe: TET-AGAAGCAGCAGTAATT-MGBNFQ Streptococcus mitis F9 primer: GGAAATTACACGTGTAGGA R9 primer: TCTAACTAAGGGTTTTCAGTCA P9 probe: CCATGTTGTCATGATGGGACGATA Streptococcus mutans F10 primer: AGAGGAATATACCAATGTTGTG R10 primer: AAGAACCATCTGTTGAAGAC P10 probe: TGTGGATAAGTTTGCGGAATGGG Streptococcus sanguis F11 primer: GACTGATGAGAAGATTACCA R11 primer: TGTTGAGATATTCGTTGCTA P11 probe: GGTCAGCTAAACACTTCAACGGTACC Streptococcus salivarius F12 primer: GGGAAGCATTATGGATTACC R12 primer: ACACATCAAGGACTGACTTATC P12 probe: ATGCAACATGCTGTGGCCTTAATGA Streptococcus gallolyticus F13 primer: AAAGCTGGTGAGGACCAAGC R13 primer: GAGCGTCATCAAATCATTCA P13 probe: GCAGATACGTTGACCAATGCTTATG Streptococcus pyogenes F14 primer: AATATCTTCTGGAATCTTAGTGA R14 primer: CTTATGTCGCTAATACCAATG P14 probe: CACCCAGAGATCGGAGAAAATCTGGA Streptococcus agalactiae F15 primer: GCAAAAGAACAGATGGAACAAAGTG R15 primer: CCCCTTACTTCCTTTTTCTATACC P15 probe: CTGTTGCTACTTACGGCGATTATGG MecA F16 primer: GTAGAAATGACTGAACGTCCGATAA R16 primer: AACTTCCACATACCATCTTCT P16 probe: ACGTTGCGATCAATGTTACCGTAGT MecC F17 primer: ATAGATGCTAGAGTACAAGA R17 primer: TTTTGGTTGTAATGCTGTA P17 probe: CCAGATCCATCGTCATTTTTC Internal standard F18 primer: TTGCTTGCGGGTGAGAGT R18 primer: CGCCAACAAGCTGATAGGAC P18 probe: ATGTGCCGGGCTCCTGCAT Composition of the kit and detection method: The components of the kit for detecting the pathogens of infective endocarditis are shown in the table below.

[0016] Component name Ingredients Primer probe mixture Combinations of primers and probes provided in the examples PCR premix (this component is purchased from outside, the reagent name is PerfeCTa MultiplexqPCR ToughMix) <![CDATA[Solution containing deoxynucleoside triphosphate (dN(U)TP), MgCl2, DNA polymerase (taq enzyme), and UDG]]> Internal standard Bacterial solution containing internal standard gene fragment Positive quality control Inactivated engineered bacteria containing target gene fragments Negative control water Preparation of primer-probe mixture Primers F1-F18 and R1-R18, and probes P1-P18 were synthesized by Nanjing General Biotechnology Co., Ltd. The probes were labeled with different fluorescent dyes: P1 was labeled with FAM and JOE, respectively, and designated P1-FAM and P1-JOE. P2 was labeled with FAM and designated P2-FAM. P3 was labeled with FAM and designated P3-FAM. P4 was labeled with JOE and designated P4-JOE. P5 was labeled with JOE and designated P5-JOE. P6 was labeled with CY3 and designated P6-CY3. P7 was labeled with CY5 and CY55, respectively, and designated P7-CY5 and P7-CY55. P8 was labeled with FAM and JOE, respectively, and designated P8-FAM and P8-JOE. P9 was labeled with ROX and designated P9-ROX. P10 is labeled with ROX fluorescent dye and is labeled as P10-ROX. P11 is labeled with ROX fluorescent dye and is labeled as P11-ROX. P12 is labeled with CY3 fluorescent dye and is labeled as P12-CY3. P13 is labeled with CY3 fluorescent dye and is labeled as P13-CY3. P14 is labeled with CY5 and CY55 fluorescent dyes, respectively, and is labeled as P14-CY5 and P14-CY55. P15 is labeled with CY5 fluorescent dye and is labeled as P15-CY5. P16 is labeled with CY55 fluorescent dye and is labeled as P16-CY55. P17 is labeled with CY55 fluorescent dye and is labeled as P17-CY55.

[0017] P18 was labeled with CY5 and CY55 fluorescent dyes, respectively, and labeled as P18-CY5 and P18-CY55.

[0018] Dilute with TE to 100 μM / μl, add 2 μl each of primers F1-F18 and R1-R18, totaling 72 μl, to prepare a primer mixture; refer to the table below to add different volumes of probes to prepare a primer-probe mixture.

[0019] Component name concentration Addition volume (μl) Primer mix - 72 P1-FAM 100um / ul 1.5 P1-JOE 100um / ul 1.5 P2-FAM 100um / ul 1 P3-FAM 100um / ul 0.5 P4-JOE 100um / ul 1 P5-JOE 100um / ul 2 P6-CY3 100um / ul 1.7 P7-CY5 100um / ul 1 P7-CY55 100um / ul 1 P8-FAM 100um / ul 1 P8-JOE 100um / ul 1 P9-ROX 100um / ul 0.5 P10-ROX 100um / ul 1 P11-ROX 100um / ul 1.7 P12-CY3 100um / ul 1 P13-CY3 100um / ul 0.5 P14-CY5 100um / ul 1 P14-CY55 100um / ul 0.5 P15-CY5 100um / ul 0.5 P16-CY55 100um / ul 1 P17-CY55 100um / ul 0.5 P18-CY5 100um / ul 1 P18-CY55 100um / ul 1 TE - 4.6 TOTAL 100 Methods for detecting infective endocarditis 1. Extraction: The kit of the present invention does not contain DNA / RNA extraction reagents. The reagent used is the QIAamp UCP Pathogen Mini Kit extraction kit, product number 50214.

[0020] (1) Sample to be tested: Mix 380ul of the sample to be tested and 20ul of the internal standard solution, and extract nucleic acid according to the instructions of the QIAamp UCPPathogen Mini Kit.

[0021] (2) Quality control products: Take 50ul of positive quality control product and negative quality control product and place them in 1.5 mL centrifuge tubes respectively. Add 20μL of internal standard solution and 330ul of water respectively and mix thoroughly. Refer to the instruction manual of the QIAamp UCP Pathogen Mini Kit for nucleic acid extraction.

[0022] 2. Amplification: Construction of PCR system, components are shown in the table below:

[0023] Materials: naica® fully automated microdroplet chip digital PCR system, Sapphire chip, pipette, DNase- and RNase-free centrifuge tubes, and DNase- and RNase-free pipette filter tips.

[0024] Detection method: Place the Sapphire chip steadily on the laboratory bench, gently rotate the white cover 1 / 4 turn, discard the white cover, pipette 25μl of reaction solution into the wells of the Sapphire chip, let it stand for 2-3 minutes, and then cover with the long white cover.

[0025] Turn on the Naica Geode droplet generation and amplification system and the air pump. Ensure that the air pump output pressure and the droplet generation system input pressure are stable at 1150 ± 50 mbar and do not exceed 1300 mbar.

[0026] Set up the reaction program. Place the sapphire chip on the heating block and close the Naica Geode lid. Click Run. After confirming the program is correct, click Play to begin droplet generation and the PCR reaction. After amplification is complete, read the signal.

[0027]

[0028] Click Run, and then click Play in the Run menu to run the program immediately.

[0029]

Test kit accuracy verification

[0030] At the same time, the PCR sequencing method was used to sequence the target sequence target region of these samples. The results showed that the detection results of this method were highly consistent with those obtained by the PCR sequencing method. This confirms the high detection accuracy of this scheme.

[0031]

Kit sensitivity analysis

[0032]

Kit specificity analysis

[0033] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A kit for detecting infective endocarditis pathogens based on a digital PCR platform, characterized in that: The components include: primer probe mixture, PCR premix, bacterial solution containing internal standard sequence fragments, positive quality control products and negative quality control products of inactivated engineered bacteria containing target gene fragments, The primers and probes are: Staphylococcus aureus F1 primer: 5'- AGA GCG TGC TGG ATT TGT AT -3' sequence 1 R1 primer: 5'- GCC ATC ATT TGA TCA CGA CG -3' sequence 2 P1 probe: 5'- GTC ATT CAA TTC CTG TTG CAA C -3' (MGB) sequence 3 Staphylococcus epidermidis F2 primer: CATATTACGTAGGTCAAGTTC sequence 4 R2 primer: GGTAAAGTGTAATGAAACCAG sequence 5 P2 probe: GCTCATCTGATACGCTACCGAATAC sequence 6 hemolytic Staphylococci F3 primer: AAACAAACTATGGAAATCCATC sequence 7 R3 primer: ATATCTTCAGGTACACTATCTAAG sequence 8 P3 probe: TTCTGCAGTTGAGGGAACAGATCT sequence 9 Staphylococcus hominis F4 primer: TAGATGGATCTGAAACAGTAGTAT sequence 10 R4 primer: CCTTCAACAATACCAAATTCGTC sequence 11 P4 probe: AGGTGCTTCATGTACTACAAACTCATTG sequence 12 Staphylococcus capitis F5 primer: TCAGATATTCAAACTGCAGTACG sequence 13 R5 primer: CTACTTCACCTTTTTCTTCAGA sequence 14 P5 probe: ATGGTGGCGGTCACTTAAACCA sequence 15 Enterococcus faecium F6 primer: TAGAGACATTGAATATGCC sequence 16 R6 primer: TCGAATGTGCTACAATC sequence 17 P6 probe: 5'-CGTATTGACCAGTGCATGT-3' (MGB) sequence 18 Enterococcus faecalis F7 primer: GAA AGA TGT CGC TTT CTA TGA TTA TGA sequence 19 R7 primer: GCG ACT TAA GCC ACT TCC ATC TA sequence 20 P7 probe: FAM-ATG CAA ATC CCA GCG CAT GTT CC-BHQ sequence 21 Haemophilus influenzae F8 primer: 5′-AATGCGTGATGCTGGTTATGAC sequence 22 R8 primer: 5′-AAGAGTTTTGCGATAGATTCATTGG sequence 23 P8 probe: TET-AGAAGCAGCAGTAATT-MGBNFQ sequence 24 Streptococcus mitis F9 primer: GGAAATTACACGTGTAGGA sequence 25 R9 primer: TCTAACTAAGGGTTTTCAGTCA sequence 26 P9 probe: CCATGTTGTCATGATGGGACGATA sequence 27 Streptococcus mutans F10 primer: AGAGGAATATACCAATGTTGTG sequence 28 R10 primer: AAGAACCATCTGTTGAAGAC sequence 29 P10 probe: TGTGGATAAGTTTGCGGAATGGG sequence 30 Streptococcus sanguis F11 primer: GACTGATGAGAAGATTACCA sequence 31 R11 primer: TGTTGAGATATTCGTTGCTA sequence 32 P11 probe: GGTCAGCTAAACACTTCAACGGTACC sequence 33 Streptococcus salivarius F12 primer: GGGAAGCATTATGGATTACC sequence 34 R12 primer: ACACATCAAGGACTGACTTATC sequence 35 P12 probe: ATGCAACATGCTGTGGCCTTAATGA 36 Streptococcus gallolyticus F13 primer: AAAGCTGGTGAGGACCAAGC sequence 37 R13 primer: GAGCGTCATCAAATCATTCA sequence 38 P13 probe: GCAGATACGTTGACCAATGCTTATG sequence 39 Streptococcus pyogenes F14 primer: AATATCTTCTGGAATCTTAGTGA sequence 40 R14 primer: CTTATGTCGCTAATACCAATG sequence 41 P14 probe: CACCCAGAGATCGGAGAAAATCTGGA sequence 42 Streptococcus agalactiae F15 primer: GCAAAAGAACAGATGGAACAAAGTG sequence 43 R15 primer: CCCCTTACTTCCTTTTTCTATACC sequence 44 P15 probe: CTGTTGCTACTTACGGCGATTATGG sequence 45 Methicillin-resistant gene MecA F16 primer: GTAGAAATGACTGAACGTCCGATAA sequence 46 R16 primer: AACTTCCACATACCATCTTCT sequence 47 P16 probe: ACGTTGCGATCAATGTTACCGTAGT sequence 48 Methicillin-resistant gene MecC F17 primer: ATAGATGCTAGAGTACAAGA sequence 49 R17 primer: TTTTGGTTGTAATGCTGTA sequence 50 P17 probe: CCAGATCCATCGTCATTTTTC sequence 51.

2. The infective endocarditis pathogen detection kit based on the digital PCR platform according to claim 1, characterized in that: The internal standard primers and probes, F18 primer: TTGCTTGCGGGTGAGAGT sequence 52 R18 primer: CGCCAACAAGCTGATAGGAC sequence 53 P18 probe: ATGTGCCGGGCTCCTGCAT sequence 54.

3. The infective endocarditis pathogen detection kit based on the digital PCR platform according to claim 1, characterized in that: The volume ratio of the primer-probe mixture and the PCR premix is 2-5:2-5, preferably 5:5; the volume ratio of the bacterial solution containing the internal standard sequence fragment, the positive quality control product of the inactivated engineered bacteria containing the target gene fragment, and the negative quality control product is: 10-50:10-300:10-300, preferably 20:50:

50.

4. The infective endocarditis pathogen detection kit based on the digital PCR platform according to claim 1, characterized in that: The PCR premix solution is a solution containing deoxynucleoside triphosphate (dN(U)TP), MgCl2, DNA polymerase (Taq enzyme), UDG, and 10*buffer.

5. The infective endocarditis pathogen detection kit based on the digital PCR platform according to claim 1, characterized in that: The negative control material is water.

6. The infective endocarditis pathogen detection kit based on the digital PCR platform according to claim 1, characterized in that: The target gene fragment includes: Staphylococcus aureus sequence: AGAGCGTGCTGGATTTGTATCAAAAACAGATGATTATTTTTATAACTTTATTGACACATATGGAGATAAAGTATTAGTACCATTAGCATATATTGACCTTGATGAATATGTGTTAAAGTTGCAACAGGAATTGAATGACAAAGAAAATCGTCGTGATCAAATGATGGC Staphylococcus epidermidis sequences: ATATTACGTAGGTCAAGTTCAATCTTCTGACTTACCACAACAAAGCTCATCTGATACGCTACCGAATACTGGTATGAAAGATAAAAATTTCAATACAAATGGTATAATAAGTCTATTTTTACTCACTGCTGGTTTCATTACACTTTACC Staphylococcus haemolyticus sequence: AAACAAACTATGGAAATCCATCATGACAAACATCACAACACTTATGTTACCAAATTAAATTCTGCAGTTGAGGGAACAGATCTTGAATCTAAATCAATTGAAGAAATTGTTGCTAACTTAGATAGTGTACCTGAAGATAT Staphylococcus aureus sequences TAGATGGATCTGAAACAGTAGTATCAGGTGCTTCATGTACTACAAACTCATTAGCACCAGTTGCTAAAGTTTTAAATGACGAATTTGGTATTGTTGAAGG Staphylococcus capitis sequence: TCAGATATTCAAACTGCAGTACGTAATAATGGTGGCGGTCACTTAAACCACTCATTATTCTGGGAATTATTATCACCAAATTCTGAAGAAAAAGGTGAAGTAG Enterococcus faecium sequence: AGAGCGTGCTGGATTTGTATCAAAAACAGATGATTATTTTTATAACTTTATTGACACATATGGAGATAAAGTATTAGTACCATTAGCATATATTGACCTTGATGAATATGTGTTAAAGTTGCAACAGGAATTGAATGACAAAGAAAATCGTCGTGATCAAATGATGGC Enterococcus faecalis sequence: GAAAGATGTCGCTTTCTATGATTATGATGCAAAATACATCAATAACACGATTGAAATGCAAATCCCAGCGCATGTTCCAGAAGAAGTAGCTCATCAAGCGCAAGAATACGCTAAAAAAGCGTATATTATGTTAGATGGAAGTGGCTTAAGTCGC Haemophilus influenzae sequences: AATGCGTGATGCTGGTTATGACGATGATATTTGCGGAGGAATTACTGCTGCTTCTTGTATTATTGGGCCATTAGTTCCACCGAGTATTGCAATGATTATTTACGGTGT(C / A)ATCCCAATGAATCTATCGCAAAACTCTT Streptococcus mitis sequence: GGAAATTACACGTGTAGGAGCAGATATCAAAATCAAATGTAGCAATTGTGACCATGTTGTCATGATGGGACGATATGATTTTGATCGAAAAATGAATAAAATTATTGACTGAAAACCCTTAGTTAGA Streptococcus mutans sequence: AGAGGAATATACCAATGTTGTGATTGCTAAGAATGTGGATAAGTTTGCGGAATGGGGTGTCACAGATTTTGAAATGGCACCGCAGTATGTGTCTTCAACAGATGGTTCTT Streptococcus sanguis sequence GACTGATGAGAAGATTACCAAGTGGTCAGCTAAACACTTCAACGGTACCAATATCTTAGGTCGTGGTGCTTACTATGTTCTTAAAGACTGGGCTAGCAACGAATATCTCAACA Streptococcus salivarius sequence: GGGAAGCATTATGGATTACCCTTATGCAACATGCTGTGGCCTTAATGACGAAGTGGCACATGCTTTTCCACGTCACTATATTTTGAAAGATGGTGACCTCTTGAAAGTTGATATGGTCTTGTCAGAACCTATCGATAAGTCAGTCCTTGATGTGT Gallolytic Streptococcus: AAAGCTGGTGAGGACCAAGCACTCAACCAAAAACGTGATAAATTGCTTAATCATAAAAACATTGCAGATACGTTGACCAATGCTTAGTCATGCTTGATGACGAAGAATTTTCAAGTCTATCTAACATTCGTTCAGCCATGAATGATTTGATGACGCTC Streptococcus pyogenes sequence: CTTATGTCGCTAATACCAATGCGGCTTTAGAAAAACACCCAGAGATCGGAGAAAATCTGGAGGAACTATTGGCAGATGTCACTAAGATTCCAGAAGATATT Streptococcus agalactiae sequence: GCAAAAGAACAGATGGAACAAAGTGGTTCAAAGTTCTTAGGTATTATTCTTAATAAAGTTAATGAATCTGTTGCTACTTACGGCGATTATGGAAATTACGGAAAAAGGTATAGAAAAAGGAAGTAAGGGG Methicillin-resistant gene mecA sequence: GTAGAAATGACTGAACGTCCGATAAAAATATATAATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAACGAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGTTAAAGAAGATGGTATGTGGAAGTT Methicillin-resistant gene mecC sequence: ATAGATGCTAGAGTACAAGAAAGTATTTATAAACATATGAAAAATGACGATGGATCTGGTACAGCATTACAACCAAAAACTG.

7. The infective endocarditis pathogen detection kit based on the digital PCR platform according to claim 1, characterized in that: The sequence of the internal standard: TTGCTTGCGGGTGAGAGTGGTATTGAAGTAGTTGCAGTAAACGACTTAACAGACGACGATGTGCCGGGCTCCTGCATAAATATGACACTATGCAAGGGTCCTATCAGCTTGTTGGCG.

8. Use of the kit according to claim 1 in the detection of infective endocarditis pathogens based on a digital PCR platform.

9. The detection method of the infective endocarditis pathogen detection kit based on the digital PCR platform according to any one of claims 1 to 8, characterized in that: (1) Extraction: The kit does not include DNA / RNA extraction reagents; Test sample: Take the test sample and internal standard solution and mix them for nucleic acid extraction; Quality control products: Take the positive quality control product and the negative quality control product, add the internal standard solution and water respectively, mix thoroughly, and perform nucleic acid extraction; (2) Amplification: PCR system: (3) Reaction procedure: 。

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