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WO2025002319A1 - Composition et kit pour la détection conjointe et la différenciation de pathogènes responsables d'infections sanguines, et utilisation - Google Patents

Composition et kit pour la détection conjointe et la différenciation de pathogènes responsables d'infections sanguines, et utilisation Download PDF

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Publication number
WO2025002319A1
WO2025002319A1 PCT/CN2024/102287 CN2024102287W WO2025002319A1 WO 2025002319 A1 WO2025002319 A1 WO 2025002319A1 CN 2024102287 W CN2024102287 W CN 2024102287W WO 2025002319 A1 WO2025002319 A1 WO 2025002319A1
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seq
primer
probe
upstream
downstream
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Chinese (zh)
Inventor
戴立忠
符小玉
张登峰
钟秋梅
丁峰
吴康
刘佳
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Sansure Biotech Inc
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Sansure Biotech Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/425—Serratia
    • C12R2001/43—Serratia marcescens
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the field of molecular biological detection, and specifically, relates to the detection of pathogenic bacteria related to bloodstream infection, and more specifically, to the detection of Burkholderia cepacia, Serratia marcescens and Acinetobacter baumannii.
  • Burkholderia cepacia is one of several important conditional pathogens of hospital-acquired infections. It is usually not pathogenic to healthy humans, but often invades immunocompromised individuals. It often colonizes the lungs of patients with cystic fibrosis and chronic granulomatous diseases. It is a highly virulent and lethal pathogen. In recent years, the clinical isolation rate of this bacterium has continued to increase, and it has a high resistance to a variety of antibiotics, making clinical treatment difficult.
  • This bacterium is an aerobic, non-fermenting, Gram-negative, non-spore-forming bacillus, commonly found in moist soil, water, plants, etc. It can survive and grow in nutrient-deficient water.
  • Burkholderia cepacia has special adhesins and is transmitted through close contact or inhalation of aerosols carrying the bacteria. It often attacks individuals with low immunity, especially in patients in the intensive care unit (ICU). ICU patients often have symptoms such as long-term bed rest, old age, low immune function, and impaired consciousness. Normal physiological reflexes such as swallowing and coughing are weakened or disappear to varying degrees. Sputum, blood, and vomitus are not easy to expel.
  • Burkholderia cepacia infection In addition, repeated invasive respiratory operations such as tracheotomy, endotracheal intubation, and ventilator use are all risk factors for Burkholderia cepacia infection.
  • the main symptoms caused by Burkholderia cepacia infection are bacteremia, urinary tract infection, suppurative arthritis, endocarditis, meningitis, and respiratory tract infection.
  • Serratia marcescens is an important conditional pathogen of nosocomial infection. The smallest of them is about 0.5 ⁇ (0.5 ⁇ 1.0) microns. It is a short, nearly spherical rod, but with various shapes. It is Gram-negative, has flagella all over its body, and can move. It has no capsule and spores, and about half of the strains can produce red prodigiosin. It is widely distributed in nature and is a common flora in water and soil. It often adheres to the ward environment, medical equipment, implanted catheters and the hands of medical staff.
  • the bacteria can cause sepsis, bacteremia, bloodstream infection, meningitis, endocarditis, myocarditis, pneumonia, cellulitis, wound infection, peritonitis, keratitis, endophthalmitis, urinary tract infection, empyema, cholecystitis, arthritis, osteomyelitis, necrotizing fasciitis, septic shock, suppurative thyroiditis, empyema, epidural abscess, chorioamnionitis, abscess and other diseases.
  • Serratia marcescens is naturally resistant to a variety of drugs such as ampicillin, amoxicillin, macrolides, first-generation cephalosporins, second-generation cephalosporins, cephamycins, etc., and has strong vitality, making the infections it causes more frequent and clinical treatment more difficult.
  • drugs such as ampicillin, amoxicillin, macrolides, first-generation cephalosporins, second-generation cephalosporins, cephamycins, etc.
  • carbapenem antibiotics such as meropenem, ertapenem, etc.
  • Acinetobacter baumannii is the most common Gram-negative bacillus in the genus Acinetobacter. It is widely found in water and soil in nature, hospital environment, human skin, respiratory tract, digestive tract and urogenital tract, and is a conditional pathogen. The bacterium is widely distributed in the hospital environment and can survive for a long time, which can easily cause infection in critically ill patients. Therefore, it is often isolated from specimens such as blood, urine, pus and respiratory secretions of infected patients. Among non-fermenting bacteria, the infection is second only to Pseudomonas. The infection of Acinetobacter baumannii is gradually increasing, and its drug resistance is becoming more and more serious, which has attracted serious attention from clinicians and microbiologists.
  • Acinetobacter baumannii mainly causes respiratory tract infections, and can also cause sepsis, urinary tract infections, secondary meningitis, etc. Acinetobacter baumannii is widely distributed in the hospital environment and can survive for a long time, which poses a great threat to critically ill patients and patients in CCU and ICU. This type of infection is also called ICU-acquired infection.
  • Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii can all cause sepsis or bacteremia, which are life-threatening. Therefore, early diagnosis of pathogens and timely and effective anti-infection treatment are the key to improving prognosis and can greatly reduce morbidity and mortality. It is difficult to identify the type of bacteria that cause infection through laboratory testing, and the current bacterial culture conditions are relatively harsh, resulting in a low culture positivity rate. In addition, the culture of multiple mixed infections is extremely difficult, which brings great trouble to infected patients and clinicians.
  • the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:
  • a primer set for detecting Burkholderia cepacia comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, a primer pair consisting of an upstream primer as shown in SEQ ID NO: 10 and a downstream primer as shown in SEQ ID NO: 11, and a primer pair consisting of an upstream primer as shown in SEQ ID NO: 13 and a downstream primer as shown in SEQ ID NO: 14, and the probe comprises a probe as shown in at least one sequence among SEQ ID NO: 3, SEQ ID NO: 12 and SEQ ID NO: 15;
  • a primer set for detecting Serratia marcescens comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5, a primer pair consisting of an upstream primer as shown in SEQ ID NO:16 and a downstream primer as shown in SEQ ID NO:17, and a primer pair consisting of an upstream primer as shown in SEQ ID NO:19 and a downstream primer as shown in SEQ ID NO:20, and the probe comprises a probe as shown in at least one sequence among SEQ ID NO:6, SEQ ID NO:18 and SEQ ID NO:21; and
  • a primer set for detecting Acinetobacter baumannii comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO:7 and a downstream primer as shown in SEQ ID NO:8, a primer pair consisting of an upstream primer as shown in SEQ ID NO:22 and a downstream primer as shown in SEQ ID NO:23, and a primer pair consisting of an upstream primer as shown in SEQ ID NO:25 and a downstream primer as shown in SEQ ID NO:26, and the probe comprises at least one of SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25. At least one of the probes shown in NO:27.
  • the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:
  • An upstream primer as shown in SEQ ID NO:1, a downstream primer as shown in SEQ ID NO:2 and a probe as shown in SEQ ID NO:3 are used to detect Burkholderia cepacia;
  • An upstream primer as shown in SEQ ID NO:4, a downstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6 are used to detect Serratia marcescens;
  • the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8 and the probe shown in SEQ ID NO:9 are used to detect Acinetobacter baumannii.
  • the present disclosure provides a use of the above-mentioned composition in preparing a kit for joint detection and differentiation of bloodstream infection pathogens, wherein the pathogens are Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii.
  • the present disclosure provides a kit for the joint detection and differentiation of bloodstream infection pathogens, the kit comprising the composition as described above.
  • the present disclosure provides a use for preparing a reagent for joint detection and differentiation of bloodstream infection pathogens, wherein the joint detection comprises the following steps:
  • step 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;
  • the joint detection composition provided by the present disclosure mainly utilizes a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii in a single-tube reaction system.
  • the composition disclosed in the present disclosure has a higher detection sensitivity of 500 copies/mL, good specificity, and more accurate detection.
  • FIG. 1a is a graph showing the test results of a composition disclosed herein.
  • FIG. 1b is a graph showing the test results of a composition disclosed herein.
  • FIG. 1c is a graph showing the test results of a composition disclosed herein.
  • FIG. 2 is a graph showing the sensitivity results of a composition disclosed in the present invention for detecting Burkholderia cepacia.
  • FIG. 3 is a graph showing the sensitivity results of a composition disclosed in the present invention for detecting Acinetobacter baumannii.
  • FIG. 4 is a graph showing the sensitivity results of a composition disclosed in the present invention for detecting Serratia marcescens.
  • FIG. 5 is a graph showing specific results of the disclosed composition.
  • FIG. 6 shows the results of gene amplification detection of the selected Burkholderia cepacia scpA-1 and Acinetobacter baumannii rlmA-1/rlmA-2 combination.
  • FIG. 7 shows the results of gene amplification detection of the selected Burkholderia cepacia scpA-1 and Serratia marcescens M60-1/M60-2 combination.
  • FIG8 shows the results of gene amplification detection of the selected Serratia marcescens M60-1 and Burkholderia cepacia scpA-1/scpA-2 combination.
  • FIG. 9 is a graph showing the detection results of the single target primer and the comparative primer of the composition disclosed in the present invention for detecting Burkholderia cepacia.
  • FIG. 10 is a graph showing the detection results of the single target primer and the comparative example primer of the composition for detecting Acinetobacter baumannii disclosed in the present invention.
  • FIG. 11 is a graph showing the detection results of the single target primer and the comparative example primer of the composition for detecting Serratia marcescens disclosed in the present invention.
  • the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:
  • a primer set for detecting Burkholderia cepacia comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, a primer pair consisting of an upstream primer as shown in SEQ ID NO: 10 and a downstream primer as shown in SEQ ID NO: 11, and a primer pair consisting of an upstream primer as shown in SEQ ID NO: 13 and a downstream primer as shown in SEQ ID NO: 14, and the probe comprises a probe as shown in at least one sequence among SEQ ID NO: 3, SEQ ID NO: 12 and SEQ ID NO: 15;
  • a primer set for detecting Serratia marcescens comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5, a primer pair consisting of an upstream primer as shown in SEQ ID NO:16 and a downstream primer as shown in SEQ ID NO:17, and a primer pair consisting of an upstream primer as shown in SEQ ID NO:19 and a downstream primer as shown in SEQ ID NO:20, and the probe comprises a probe as shown in at least one sequence among SEQ ID NO:6, SEQ ID NO:18 and SEQ ID NO:21; and
  • a primer set for detecting Acinetobacter baumannii comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO:7 and a downstream primer as shown in SEQ ID NO:8, a primer pair consisting of an upstream primer as shown in SEQ ID NO:22 and a downstream primer as shown in SEQ ID NO:23, and a primer pair consisting of an upstream primer as shown in SEQ ID NO:25 and a downstream primer as shown in SEQ ID NO:26, and the probe comprises a probe as shown in at least one of SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:27.
  • the primer set for detecting Burkholderia cepacia is selected from at least one of the following primer sets: a primer set consisting of an upstream primer as shown in SEQ ID NO: 1, a downstream primer as shown in SEQ ID NO: 2, and a probe as shown in SEQ ID NO: 3, a primer set consisting of an upstream primer as shown in SEQ ID NO: 10, a downstream primer as shown in SEQ ID NO: 11, and a probe as shown in SEQ ID NO: 12, or an upstream primer as shown in SEQ ID NO: 13, a downstream primer as shown in SEQ ID NO: 14.
  • the primer group for detecting Serratia marcescens is selected from at least one of the primer groups shown below: a primer group consisting of an upstream primer shown in SEQ ID NO:4, a downstream primer shown in SEQ ID NO:5, and a probe shown in SEQ ID NO:6, a primer group consisting of an upstream primer shown in SEQ ID NO:16, a downstream primer shown in SEQ ID NO:17, and a probe shown in SEQ ID NO:18, or a primer group consisting of an upstream primer shown in SEQ ID NO:19, a downstream primer shown in SEQ ID NO:20, and a probe shown in SEQ ID NO:21.
  • the primer set for detecting Acinetobacter baumannii is selected from at least one of the primer sets shown below: a primer set consisting of an upstream primer shown in SEQ ID NO:7, a downstream primer shown in SEQ ID NO:8, and a probe shown in SEQ ID NO:9, a primer set consisting of an upstream primer shown in SEQ ID NO:22, a downstream primer shown in SEQ ID NO:23, and a probe shown in SEQ ID NO:24, or a primer set consisting of an upstream primer shown in SEQ ID NO:25, a downstream primer shown in SEQ ID NO:26, and a probe shown in SEQ ID NO:27.
  • the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:
  • upstream primers, downstream primers and probes for detecting Acinetobacter baumannii are shown in SEQ ID NO:7 ⁇ 9.
  • the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:
  • An upstream primer as shown in SEQ ID NO:1, a downstream primer as shown in SEQ ID NO:2 and a probe as shown in SEQ ID NO:3 are used to detect Burkholderia cepacia;
  • An upstream primer as shown in SEQ ID NO:4, a downstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6 are used to detect Serratia marcescens;
  • the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8 and the probe shown in SEQ ID NO:9 are used to detect Acinetobacter baumannii.
  • the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:
  • An upstream primer as shown in SEQ ID NO:1, a downstream primer as shown in SEQ ID NO:2 and a probe as shown in SEQ ID NO:3 are used to detect Burkholderia cepacia;
  • An upstream primer as shown in SEQ ID NO:4, a downstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6 are used to detect Serratia marcescens;
  • the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8 and the probe shown in SEQ ID NO:9 are used to detect Acinetobacter baumannii.
  • the joint detection composition provided by the present disclosure mainly uses a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously detecting and distinguishing Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii in a single-tube reaction system.
  • the composition disclosed in the present disclosure has a higher detection sensitivity of 500 copies/mL, good specificity, and more accurate detection.
  • composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.
  • the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is RNase P.
  • the probes in the composition are labeled with a fluorescent reporter group at the 5' end.
  • the fluorescent reporter groups of the probes of the disclosed composition are different from each other and do not interfere with each other.
  • fluorescent reporter groups used by each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection.
  • different channels can be used for detection.
  • ATTO 425, Quasar 705, FAM, HEX, ROX and CY5 can be used.
  • the absorbance values of these groups are not close, and different channels can be selected, so they will not interfere with each other.
  • the fluorescent reporter group of the Burkholderia cepacia probe is FAM; the fluorescent reporter group of the Acinetobacter baumannii probe is HEX (or VIC); and the fluorescent reporter group of the Serratia marcescens probe is ROX.
  • composition of the present disclosure may simultaneously include one or more of the primer sets consisting of the above primer pairs and probes.
  • primer set refers to upstream and downstream primers and probes that match each other for detecting a target.
  • compositions disclosed herein can be combined into any combination of corresponding target detection forms. Those skilled in the art can combine as needed to detect which targets, that is, to combine primers and probe pairs corresponding to the target. These combinations are all included in the present disclosure.
  • compositions are used for fluorescent PCR.
  • the 3' end of the probe also has a non-fluorescent quencher, such as NFQ and QSY.
  • the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.
  • the 3' end of the probe is BHQ1.
  • each component of the composition of the present disclosure is present in a separate package.
  • the components of the composition of the present disclosure are present in the same package.
  • composition of the present disclosure are present in a mixed form.
  • the present disclosure provides a use of the above-mentioned composition of the present disclosure in the preparation of a kit for joint detection and differentiation of bloodstream infection pathogens, wherein the pathogens are Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii.
  • the present disclosure provides a kit for the joint detection and differentiation of bloodstream infection pathogens, the kit comprising the composition of the present disclosure as described above.
  • the kit also includes a negative quality control product and a positive quality control product.
  • the negative control is at least one of DEPC H 2 O, physiological saline, and an internal standard gene.
  • the positive control is at least one of a plasmid or DNA fragment of Burkholderia cepacia, Serratia marcescens, or Acinetobacter baumannii.
  • the kit also includes at least one of dNTP, PCR buffer and Mg 2+ .
  • the kit also includes: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a DNA polymerase.
  • the kit also includes at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTP, dUTP, uracil glycosylase (UDG), a DNA polymerase, a PCR buffer and Mg 2+ .
  • the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.
  • the disclosed kit includes Taq enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.
  • PCR buffers are composed of Tris-HCl, MgCl 2 , KCl, Triton X-100, etc.
  • the total volume in a single PCR reaction tube is generally 20 ⁇ l to 200 ⁇ l.
  • the kit disclosed herein is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.
  • a reagent for preparing a bloodstream infection pathogen joint detection and differentiation comprising the following steps:
  • step 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;
  • the present disclosure also provides a method for joint detection and differentiation of bloodstream infection pathogens, the method comprising the following steps:
  • step 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;
  • the method can be used for the diagnosis and treatment of a disease, can be used for auxiliary diagnosis of a disease, or can be used for neither diagnosis nor treatment of a disease.
  • the method is not used for the diagnosis of a disease, that is, non-diagnostic purpose.
  • non-diagnostic purpose means that the method is not intended to obtain whether an individual is infected with the above-mentioned pathogens and suffers from bloodstream infection.
  • the method can be used to detect the presence of the above pathogens in a test culture (such as blood).
  • a method for joint detection and differentiation of bloodstream infection pathogens for non-diagnostic purposes comprising the following steps:
  • step 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition disclosed above or the kit disclosed above;
  • the sample used for detection may be blood, plasma, etc., but is not limited thereto. It may also be alveolar lavage fluid, sputum, punctured tissue, pus, wound secretions, and the like.
  • reaction conditions of the fluorescent quantitative PCR are: DNA pre-denaturation at 95°C for 1 to 3 minutes, 1 cycle; denaturation at 95°C for 5 to 20 seconds, annealing at 55°C to 60°C for 10 to 60 seconds, 30 to 50 cycles, and fluorescence collection.
  • the primers and probes used in the present disclosure are shown in Table 1. Sequences with a similarity of more than 80% to the sequences (SEQ ID NO: 1 to SEQ ID NO: 9) can be amplified.
  • the fluorescent reporter group of the Burkholderia cepacia scpA probe is FAM
  • the fluorescent reporter group of the Serratia marcescens M60 probe is ROX
  • the fluorescent reporter group of the Acinetobacter baumannii rlmA probe is HEX.
  • Example 2 Method for detecting pathogens
  • Fluorescence PCR amplification reaction solution contains PCR buffer, Taq enzyme, Mg 2+ , dNTP, primers, probes, etc.
  • the specific reaction system is shown in Table 2.
  • the results are automatically saved, and the amplification curves of the detection targets are analyzed separately.
  • adjust the Start value, End value, and Threshold value of the Baseline users can adjust according to actual conditions, the Start value can be set between 3 and 15, and the End value can be set between 5 and 20, and adjust the amplification curve of the negative control to make it flat or below the threshold line
  • click Analyze to analyze, so that each parameter meets the requirements of the following "Quality Control", and then record the qualitative results in the Plate window.
  • Negative control no Ct value is displayed in the curves of the three channels of FAM, ROX, and HEX;
  • the Ct reference value of the target gene detected by this kit was determined to be 38 through reference value research.
  • Example 1 The primers and probes shown in Example 1 were used to perform PCR detection on Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii on a Hongshi fluorescent quantitative PCR instrument according to the method of Example 2.
  • the detection results are shown in Figures 1a to 1c. It can be seen from the figures that the compositions disclosed in the present invention can perform good detection on various pathogens. Although the fluorescence intensity of the compositions provided by SEQ ID NO: 10 to SEQ ID NO: 27 is relatively weak, they can also perform good discrimination on various pathogens.
  • Example 1 of the present disclosure Using the composition in Example 1 of the present disclosure (scpA group corresponding to SEQ ID NO: 1 to SEQ ID NO: 3, rlmA group corresponding to SEQ ID NO: 6 to SEQ ID NO: 9, and M60 group corresponding to SEQ ID NO: 4 to SEQ ID NO: 6), LOD (sensitivity) detection was performed on each target (corresponding to Burkholderia cepacia scpA, Acinetobacter baumannii rlmA, and Serratia marcescens M60, respectively) to simulate clinical samples and perform multiplex PCR detection on a Macrostone fluorescent quantitative PCR instrument. The results are shown in Figures 2 to 4, indicating that each channel can still accurately detect samples as low as 500 copies/mL, indicating that the sensitivity of the disclosed composition is high, and the detection limit is ⁇ 500 copies/mL.
  • Example 1 of the present disclosure In order to test the blank specificity of the composition in Example 1 of the present disclosure (scpA group corresponding to SEQ ID NO: 1 to SEQ ID NO: 3, rlmA group corresponding to SEQ ID NO: 6 to SEQ ID NO: 9, and M60 group corresponding to SEQ ID NO: 4 to SEQ ID NO: 6), the negative control was used as a sample and the above-mentioned operation steps were followed for detection. As shown in Figure 5, there was no non-specific amplification in each target channel, and the blank specificity of the kit was good.
  • Comparative Example 1 Other primers and probes designed by the present disclosure that have poor effects
  • dimers may form between primers and (or) probes, but this probability is very small and can be excluded at the beginning of the design.
  • dimers are likely to occur between primers and primers, probes and probes, or primers and probes.
  • the conservatism of the design conservatism is crucial to the accuracy of the detection) and the mutual interference between different primers and probes must be considered, so the primers and probes need to be carefully designed.
  • the inventors also designed other primers and probes to form different detection systems 1 to 3 (sequences not shown), which are also used to detect the above pathogens. Specific detection results are shown in Figures 6 to 8.
  • the inventors also compared the amplification effects for each target, and the results are shown in Figures 9 to 11.
  • the remaining primers and probes have a smaller gap in the detection effect of a single target, but in the system of joint inspection, the gap in the amplification effect is significantly larger than that of a single target detection, which may be due to the influence of primers and probes on each other in the joint inspection system; specifically, as shown in Figure 9, the primer groups scpA-1 and scpA-2 designed for Burkholderia cepacia, respectively, have similar detection effects, but Burkholderia cepacia scpA-1 and scpA-2 are mixed with Serratia marcescens primer group M60-1 for amplification detection, and the combination of M60-1 and scpA-1 is significantly better than M60-1 and scpA-2. Therefore, it is shown that the joint inspection system disclosed in the present invention has superiority.

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Abstract

La présente invention concerne une composition et un kit pour la détection conjointe et la différenciation d'agents pathogènes responsables d'infections sanguines, ainsi qu'une utilisation. Selon la composition proposée pour la détection conjointe, en utilisant principalement un procédé d'analyse par PCR à fluorescence multiple, différents agents pathogènes sont détectés en détectant des cibles sur différents agents pathogènes, de sorte que la détection et la différenciation de Burkholderia cepacia, Serratia marcescens et Acinetobacter baumannii sont toutes deux réalisées dans un système de réaction monotube.
PCT/CN2024/102287 2023-06-28 2024-06-28 Composition et kit pour la détection conjointe et la différenciation de pathogènes responsables d'infections sanguines, et utilisation Pending WO2025002319A1 (fr)

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CN116732210B (zh) * 2023-08-08 2024-07-09 圣湘生物科技股份有限公司 检测败血病相关病原体的组合物、试剂盒及用途

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