CN116875716A - Composition, kit and application for joint detection and differentiation of blood stream infection pathogens - Google Patents
Composition, kit and application for joint detection and differentiation of blood stream infection pathogens Download PDFInfo
- Publication number
- CN116875716A CN116875716A CN202310959442.8A CN202310959442A CN116875716A CN 116875716 A CN116875716 A CN 116875716A CN 202310959442 A CN202310959442 A CN 202310959442A CN 116875716 A CN116875716 A CN 116875716A
- Authority
- CN
- China
- Prior art keywords
- composition
- streptococcus
- kit
- detection
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 244000052769 pathogen Species 0.000 title claims abstract description 21
- 230000004069 differentiation Effects 0.000 title claims abstract description 13
- 206010040047 Sepsis Diseases 0.000 title description 4
- 208000037815 bloodstream infection Diseases 0.000 title description 2
- 241000194017 Streptococcus Species 0.000 claims abstract description 41
- 241000194031 Enterococcus faecium Species 0.000 claims abstract description 31
- 241001134658 Streptococcus mitis Species 0.000 claims abstract description 31
- 206010002383 Angina Pectoris Diseases 0.000 claims abstract description 28
- 208000015181 infectious disease Diseases 0.000 claims abstract description 19
- 230000017531 blood circulation Effects 0.000 claims abstract description 10
- 239000000523 sample Substances 0.000 claims description 52
- 210000004369 blood Anatomy 0.000 claims description 11
- 239000008280 blood Substances 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 10
- 125000006853 reporter group Chemical group 0.000 claims description 9
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 238000003908 quality control method Methods 0.000 claims description 8
- 230000001717 pathogenic effect Effects 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 239000012678 infectious agent Substances 0.000 claims description 6
- 238000003753 real-time PCR Methods 0.000 claims description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 5
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 15
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 238000007689 inspection Methods 0.000 abstract description 5
- 238000010222 PCR analysis Methods 0.000 abstract description 2
- 244000052616 bacterial pathogen Species 0.000 abstract description 2
- 238000011269 treatment regimen Methods 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 description 12
- 238000003199 nucleic acid amplification method Methods 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 8
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 101100178983 Caenorhabditis elegans hyl-1 gene Proteins 0.000 description 4
- 101100178984 Caenorhabditis elegans hyl-2 gene Proteins 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241001478240 Coccus Species 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 208000022362 bacterial infectious disease Diseases 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000007847 digital PCR Methods 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 208000013223 septicemia Diseases 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 description 1
- WNDDWSAHNYBXKY-UHFFFAOYSA-N ATTO 425-2 Chemical compound CC1CC(C)(C)N(CCCC(O)=O)C2=C1C=C1C=C(C(=O)OCC)C(=O)OC1=C2 WNDDWSAHNYBXKY-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000194008 Streptococcus anginosus Species 0.000 description 1
- 241000960363 Streptococcus infantis Species 0.000 description 1
- 241000194019 Streptococcus mutans Species 0.000 description 1
- 208000033809 Suppuration Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 206010048038 Wound infection Diseases 0.000 description 1
- 206010048629 Wound secretion Diseases 0.000 description 1
- 206010000269 abscess Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000036626 alertness Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000005002 female reproductive tract Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000007236 host immunity Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 201000007119 infective endocarditis Diseases 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 210000003300 oropharynx Anatomy 0.000 description 1
- 206010034674 peritonitis Diseases 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 201000009890 sinusitis Diseases 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009974 thixotropic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
Classifications
-
- 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
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of molecular biology detection, and particularly relates to detection of pathogenic bacteria related to blood flow infection, and more particularly relates to detection of streptococcus angina, streptococcus mitis and enterococcus faecium. The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that detection and differentiation of streptococcus strainogenes, streptococcus mitis and enterococcus faecium are realized in a single-tube reaction system at the same time, and the treatment strategy is improved in a targeted manner. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to detection of pathogens related to blood flow infection, and more particularly relates to detection of streptococcus angina, streptococcus mitis and enterococcus faecium.
Background
The streptococcus angina (Streptococcus anginosus) is one of normal flora planted in the oral cavity, the throat, the digestive tract and the like, and is one of the streptococcus miller. Streptococcus angina is a gram-positive, catalase-negative facultative anaerobic coccus that forms microcolonies on agar medium, belonging to the group of streptococcus grass green. The most common diseases caused by streptococcus angina are respiratory tract infection, suppurative infection and blood flow infection, which account for 34.8%, 33.5% and 8.5% respectively. Previous studies have demonstrated that streptococcus angina produces a variety of toxins in vitro, which often cause invasive suppurative infections, and once invading blood, can circulate throughout the body with blood, causing systemic severe infectious diseases. The blood flow infection of the streptococcus angina group is mostly related to invasive operation, and most patients are combined with basic diseases such as digestive system, cardiovascular system, respiratory system diseases and tumors due to clinical characteristics of the blood flow infection of the streptococcus angina. A plurality of researches show that the most common basic disease in blood flow infection of the streptococcus angina is tumor, and the occupied lesion caused by the tumor is presumed to destroy the normal structure of the human body to cause local infection, so that the streptococcus angina is infected. The streptococcus angina infection features are not obvious, the identification difficulty is high, in clinical work, if the streptococcus angina infection is suspected to be related bacterial infection, the specimen should be examined in time, antibiotics are reasonably applied according to the drug sensitivity result in time, and drug resistance monitoring is carried out so as to improve prognosis. Regarding the identification of streptococcus angina, most countries employ automatic identification instruments and API 20Strep kits, etc. Only a few big hospitals in China can adopt the method, namely 100% of the method can not be used for making correct identification, but the method is still the main identification method of a clinical laboratory.
The group of streptococcus mitis includes streptococcus mitis (Streptococcus mitis, s. Mis), streptococcus stomatus, streptococcus infantis, streptococcus mutans, streptococcus cristae, etc. The streptococcus mitis belongs to one of the streptococcus herbicola, is one of normal flora of human body, is mainly distributed at the oropharynx part, and can be seen in skin, gastrointestinal tract and female genital tract. Streptococcus mitis is gram-positive coccus, spherical or elliptic, and forms short or long chain in serum broth, and has no spore, no power, facultative anaerobic and thixotropic negative. Streptococcus mitis belongs to conditional pathogenic bacteria and can cause a series of invasive diseases, especially in patients with immunodeficiency such as malignant tumor, chemotherapy, neutrophil deficiency and the like. Streptococcus mitis can cause blood flow infection, infectious endocarditis, meningitis, pneumonia, odontogenic infection, sinusitis and the like, and can also cause abscess lesions. Streptococcus mitis has the same genetic structure encoding gentamicin resistance as enterococcus faecium and enterococcus faecium, and such genetic determinant is integrated on chromosome instead of plasmid. Infection with Streptococcus mitis is not uncommon, and its causes and clinical types are various, but frequently missed due to misidentification of Streptococcus viridis in culture. However, the development of drug resistance of streptococcus mitis is faster than that of streptococcus herbicola, so that the microbial room and clinicians can strengthen alertness and improve diagnosis level in response to the bacterial infection.
Enterococcus faecium (Enterococcus faecium) belongs to the genus enterococcus and is part of the normal flora in the human and animal intestinal tract. Enterococcus faecium is usually present in the intestinal tract of humans and animals and does not cause host disease. However, when the host immunity is reduced or the host lacks nutrition, the disease of the organism is caused, and wound infection and peritoneal infection of the organism are usually caused, and bacteremia and the like are caused. Can cause septicemia, endocarditis, encephalitis and the like in mixed infection of low immunity and excessive use of antibiotics and various pathogens, and has the death rate of 21.0-27.5 percent. Enterococcus faecium, a conditional pathogen, has been increasing in infection rates in the united states, europe and south america in recent years. The Chinese bacteria drug resistance monitoring net shows that enterococcus detection increasing year by year is mainly performed by enterococcus faecium. The method for primarily identifying the pathogen by utilizing the staining form and biochemical property of enterococcus faecium is long in time consumption and easy to confuse with other bacteria, so that a nucleic acid diagnosis method such as DNA molecular hybridization or Polymerase Chain Reaction (PCR) technology can be adopted to quickly diagnose enterococcus faecium, and has important guiding significance for early diagnosis of clinical infection.
The streptococcus angina, streptococcus mitis and enterococcus faecium can cause septicemia and endanger life safety, so that early diagnosis of pathogens and administration of timely and effective anti-infection treatment are key for improving prognosis, and the morbidity and mortality can be greatly reduced. However, it is difficult to identify the species of bacteria infected by clinical symptoms and conventional laboratory tests, and the current bacterial culture conditions are severe, resulting in low culture positive rate and extremely difficult culture of multiple mixed infections, which brings great trouble to infected patients and clinicians.
Therefore, there is a need in the art for a product that can simply and rapidly detect such pathogens in order to specifically improve therapeutic strategies, and that has high sensitivity and good specificity.
Disclosure of Invention
In view of this, in a first aspect, the present invention provides a composition for joint detection and differentiation of blood stream infectious agents comprising:
an upstream primer, a downstream primer and a probe for detecting streptococcus angina, which are shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting streptococcus mitis, which are shown in SEQ ID NO. 4-6; and an upstream primer, a downstream primer and a probe for detecting enterococcus faecium, which are shown in SEQ ID NO. 7-9.
The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that detection and differentiation of streptococcus prandis, streptococcus mitis and enterococcus faecium are simultaneously carried out in a single-tube reaction system, and a treatment strategy is improved in a targeted manner. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection.
Further, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.
In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is Rnase P.
Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.
As used herein, "distinct and non-interfering with each other" means that the fluorophores used for each probe in the composition are different and do not affect each other's detection, i.e., can be performed using different channels. For example, ATTO 425, quasar705, FAM, HEX, ROX and CY5 can be used, which groups do not have close absorbance values and can select different channels so as not to interfere with each other.
In some specific embodiments, the fluorescent reporter group of the streptococcus angina probe is FAM; the fluorescent reporter group of the streptococcus mitis probe is HEX; the fluorescent reporter group of the enterococcus faecium probe is ROX.
Further, in some embodiments, the compositions of the present invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to matched upstream and downstream primers and probes that detect a target.
The composition of the invention can be combined into any combination form for detecting 3 targets. Those skilled in the art can combine the primers and probe pairs as necessary to detect which targets are the corresponding targets. These combinations are included in the present invention.
For example, any 3 pairs of the above 3 pairs of primers and probes may be included, any 2 pairs of the above 3 pairs of primers and probes may be included, and any 1 pair of the above 3 pairs of primers and probes may be included.
In some specific embodiments, the compositions of the invention are used in fluorescent PCR.
Further, the 3' end of the probe also has a non-fluorescent quencher.
Further, the 3' -end of the probe also has a quenching group, such as BHQ1 or BHQ2.
In a specific embodiment, the 3' end of the probe is BHQ1.
In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.
In a particular embodiment, the ingredients of the composition of the invention are present in the same package.
Further, the components of the composition of the present invention are present in a mixed form.
In a second aspect, the invention provides the use of the composition of the invention described above for the preparation of a kit for joint detection and differentiation of a pathogen of blood flow infection, wherein the pathogen is streptococcus angina, streptococcus mitis, enterococcus faecium.
In a third aspect, the present invention provides a kit for joint detection and differentiation of blood stream infectious agents, said kit comprising a composition of the present invention as described above.
Further, the kit also comprises a negative quality control and a positive quality control.
In a specific embodiment, the negative quality control is DEPC H 2 O, normal saline and an internal standard gene. The positive quality control substance is at least one of segment plasmid or segment DNA of streptococcus angina, streptococcus mitis and enterococcus faecium.
Further, the kit also comprises dNTP, PCR buffer solution and Mg 2+ At least one of them.
Still further, the kit further comprises: a nucleic acid extraction reagent, and at least one of a DNA polymerase.
Further, the kit further comprises a nucleic acid extraction testAgents, dNTPs, dUTP, uracil glycosylase (UDG), DNA polymerase, PCR buffer and Mg 2+ At least one of them.
Further, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.
In a specific embodiment, the kit of the invention comprises Taq enzyme, mg 2+ dNTP (U) s, primers, probes and PCR buffer.
Common PCR buffer consists of Tris-HCl and MgCl 2 Buffer systems such as KCl and Triton X-100. The total volume in a typical single PCR reaction tube is 20. Mu.l to 200. Mu.l.
In a specific embodiment, the kit of the invention is compatible with digital PCR amplification systems, i.e., can be used directly on a digital PCR instrument for amplification.
In a fourth aspect, there is provided a method of co-detecting and differentiating blood flow-infecting pathogens for non-diagnostic purposes, the method comprising the steps of:
1) Extracting or releasing nucleic acid of a sample to be tested;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
In the present invention, the sample for detection may be blood, plasma, or the like, but is not limited thereto. But also alveolar lavage fluid, sputum, puncture tissue, pus, wound secretions, and the like.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
pre-denaturation of DNA at 95 ℃ for 1-3 min for 1 cycle; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
In a specific embodiment, there is provided the use of a composition for the preparation of a joint test and differentiation of blood stream infectious agents, said joint test comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;
3) The results were obtained and analyzed.
Further, the reaction conditions of the fluorescent quantitative PCR are as follows:
pre-denaturation of DNA at 95 ℃ for 1-3 min for 1 cycle; denaturation at 95 ℃ for 5-20 seconds, annealing at 55-60 ℃ for 10-60 seconds, 30-50 cycles, and fluorescence collection.
In this context, the term "non-diagnostic purpose" refers to information not intended to obtain whether an individual is infected with the above-mentioned pathogen and suffering from a blood flow infection. For example, the method may be used to detect the presence of the aforementioned pathogens in a test culture (e.g., blood) in need thereof.
Drawings
FIG. 1 is a graph showing the results of detection of a composition of the present invention (Streptococcus angina, streptococcus mitis, enterococcus faecium, respectively);
FIG. 2 is a graph of the sensitivity results of the compositions of the present invention (Streptococcus angina);
FIG. 3 is a graph showing the sensitivity results of the compositions of the present invention (Streptococcus mitis);
FIG. 4 is a graph of the sensitivity results of the compositions of the present invention (enterococcus faecium);
FIG. 5 is a graph of the results of the specificity of the compositions of the present invention;
FIG. 6 is a graph showing the results of single-target primer and comparative primer tests for compositions of the present invention (Streptococcus angina);
FIG. 7 is a graph showing the results of single-target primer and comparative primer tests for compositions of the present invention (Streptococcus mitis);
FIG. 8 is a graph of the results of single-target primer and comparative primer assays (enterococcus faecium) for compositions of the invention;
FIG. 9 shows the effect of amplification detection of selected combinations of the genes for Streptococcus strainogenes silB-1 and Streptococcus mitis LTA-1/LTA-2;
FIG. 10 shows the effect of amplification detection of selected combinations of genes of Streptococcus mitis LTA-1 and Streptococcus angina silB-1/silB-2;
FIG. 11 shows the effect of amplification detection of selected combinations of Streptococcus angina silB-1 and enterococcus faecium hyl-1/hyl-2.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Example 1, primers and probes used in the present invention
The primers and probes used in the present invention are shown in Table 1 below.
TABLE 1
Wherein, the fluorescent reporter group of the streptococcus angina probe is FAM; the fluorescent reporter group of the streptococcus mitis probe is HEX; the fluorescent reporter group of the enterococcus faecium probe is ROX.
Example 2 methods for detecting Streptococcus angina, streptococcus mitis and enterococcus faecium
Fluorescent PCR amplification reaction solution: contains PCR buffer solution, taq enzyme and Mg 2+ Dntps, primers, probes, and the like. The specific reaction system is shown in Table 2.
TABLE 2
| Component (A) | Volume/concentration in each reaction |
| PCR buffer | 23.5μL |
| Taq enzyme | 2.5μL(5U/μL) |
| Mg 2+ | 0.3μL(1M) |
| dNTP | 1μL |
| Primer probe | 0.25μL |
| Sample DNA | 20μL |
| ddH 2 O | Is added to 50 mu L |
The amplification reaction procedure is shown in Table 3.
TABLE 3 Table 3
Result analysis and judgment:
and after the reaction is finished, automatically storing the result, and respectively analyzing the amplification curves of the detection targets. And (3) adjusting the Start value, end value and Threshold value of Baseline according to the analyzed image (a user can adjust the Start value at 3-15, the End value can be set at 5-20, the amplification curve of the negative control is adjusted to be straight or lower than a Threshold line), clicking Analyze for analysis, so that various parameters meet the requirements in quality control, and recording qualitative results under a Plate window.
Quality control
Negative control: the curves of the FAM, ROX, HEX three channels are all without Ct values;
positive control: the curve Ct of the FAM, ROX, HEX three channels is less than or equal to 36;
the requirements are met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.
Positive judgment value
The Ct reference value of the target gene detected by the kit is determined to be 38 through the research of the reference value.
Based on the above detection results, the determination results are shown in table 4 below:
TABLE 4 Table 4
Example 3 detection results of test samples of the inventive composition
The primers and probes shown in the example 1 are used for carrying out PCR detection on streptococcus strainous, streptococcus mitis and enterococcus faecium according to the method of the example 2, the detection result is shown in the figure 1, and the composition can be used for carrying out good detection and differentiation on streptococcus strainous, streptococcus mitis and enterococcus faecium.
Example 4 sensitivity of the composition of the invention
Using the composition of example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate a clinical sample, and multiplex PCR detection was performed on a fluorescent quantitative PCR instrument. The results are shown in FIGS. 2-4, which show that each channel can still be accurately detected for samples as low as 500 copies/mL, indicating that the sensitivity of the composition of the present invention is 500 copies/mL.
EXAMPLE 5 specificity of the composition of the invention
In order to test the blank specificity of the composition of example 1 of the present invention, a negative control was used as a sample, and the test was performed according to the procedure described above. As shown in FIG. 5, each target channel has no non-specific amplification, and the blank specificity of the kit is good.
Comparative example 1, remaining poorly performing primers and probes designed according to the invention
Because of the base-pairing rules, dimers are formed between the primer and/or probe, but with little probability, this can be eliminated at the beginning of the design. However, when multiple pathogens are jointly detected, a plurality of primers and probes are arranged, dimers are easy to occur between the primers and the primers, between the probes and the probes or between the primers and the probes, so that the conservation of design (which is crucial to the accuracy of detection) is ensured, and the mutual interference among different primer probes is considered, so that the primer probes need to be carefully designed.
Therefore, the inventors also conducted comparison of amplification effects for each target, and the results are shown in FIGS. 6 to 8, and also constituted different detection systems 1 to 3 (sequences not shown) for detecting the above pathogens, and the specific detection results are shown in FIGS. 9 to 11.
As can be seen from the figure, the rest primers and probes have smaller differences in the single-target detection effect, but are placed in a joint inspection system, the differences in amplification effect are obviously larger than those of single-target detection, which is probably caused by the influence of the mutual primer probes of the joint inspection system; specifically, as shown in FIG. 8, the primer pairs hyl-1 and hyl-2 designed for enterococcus faecium respectively have close detection effects, but as shown in FIG. 11, enterococcus faecium hyl-1 and hyl-2 are mixed with streptococcus strainotis silB-1 respectively for amplification detection, and the combination of detection results hyl-1 and silB-1 is significantly better than hyl-2 and silB-1. Therefore, the joint inspection system of the invention has advantages. Similarly, the combination of the other two targets can also show similar detection results.
Claims (10)
1. A composition for joint detection and differentiation of blood stream infectious agents comprising:
an upstream primer, a downstream primer and a probe for detecting streptococcus angina, which are shown in SEQ ID NO. 1-3;
an upstream primer, a downstream primer and a probe for detecting streptococcus mitis, which are shown in SEQ ID NO. 4-6; and
an upstream primer, a downstream primer and a probe for detecting enterococcus faecium are shown in SEQ ID NO. 7-9.
2. The composition of claim 1, further comprising an upstream primer, a downstream primer, and a probe for detecting an internal standard.
3. The composition of claim 1, wherein the fluorophores of the composition probes are different from each other and do not interfere with each other.
4. The composition of claim 3, wherein the fluorescent reporter group of the streptococcus angina probe is FAM; the fluorescent reporter group of the streptococcus mitis probe is HEX; the fluorescent reporter group of the enterococcus faecium probe is ROX.
5. The composition according to any one of claims 1 to 4, wherein the components of the composition are present in a mixed form.
6. Use of a composition according to any one of claims 1 to 5 in the preparation of a kit for joint detection and differentiation of a pathogen of blood flow infection, wherein the pathogen is streptococcus angina, streptococcus mitis, enterococcus faecium.
7. A kit for joint detection and differentiation of blood stream infectious agents, said kit comprising a composition according to any one of claims 1 to 5.
8. The kit of claim 7, further comprising a negative quality control and a positive quality control.
9. The kit of claim 7 or 8, wherein the testThe kit further comprises: nucleic acid extraction reagent, DNA polymerase, dNTP, dUTP, UDG enzyme, PCR buffer and Mg 2+ At least one of them.
10. Use of a reagent for preparing a joint test and differentiation of blood stream infectious agents, said joint test comprising the steps of:
1) Extracting nucleic acid of a sample to be detected;
2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 5 or the kit of any one of claims 7 to 9;
3) The results were obtained and analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310959442.8A CN116875716A (en) | 2023-08-01 | 2023-08-01 | Composition, kit and application for joint detection and differentiation of blood stream infection pathogens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310959442.8A CN116875716A (en) | 2023-08-01 | 2023-08-01 | Composition, kit and application for joint detection and differentiation of blood stream infection pathogens |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116875716A true CN116875716A (en) | 2023-10-13 |
Family
ID=88260381
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310959442.8A Pending CN116875716A (en) | 2023-08-01 | 2023-08-01 | Composition, kit and application for joint detection and differentiation of blood stream infection pathogens |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116875716A (en) |
-
2023
- 2023-08-01 CN CN202310959442.8A patent/CN116875716A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110714090B (en) | Kit for detecting free nucleic acid of blood flow infection pathogen in blood plasma | |
| CN111004862B (en) | Primer and probe for rapidly detecting and identifying cryptococcus and application thereof | |
| CN113999933A (en) | Method for accurately detecting cryptococcus neoformans | |
| CN113512602A (en) | Blood stream infection pathogen multiple gene detection system and kit and application thereof | |
| CN113862393A (en) | Method for rapidly detecting cryptococcus gatherensis | |
| CN114134219A (en) | Multiple nucleic acid detection system and preparation method and application thereof | |
| CN114457174B (en) | Multiplex fluorescent quantitative probe method PCR kit for detecting urinary tract pathogen infection | |
| CN112680541B (en) | LNA-Taqman-multiplex fluorescence PCR technology and application thereof in rapid detection of candida | |
| CN116875716A (en) | Composition, kit and application for joint detection and differentiation of blood stream infection pathogens | |
| CN106048051B (en) | A kind of candida krusei fluorescence PCR detection reagent kit | |
| CN111455094B (en) | Composition, kit, application and method for detecting deep infection fungi | |
| CN115976240B (en) | Composition, kit, method and use for detecting sepsis pathogen | |
| CN116516038B (en) | Composition, kit and application for joint detection and differentiation of blood stream infection pathogens | |
| CN116732210A (en) | Composition for detecting septicemia related pathogens, kit and application | |
| CN118006812A (en) | Composition, kit and application for detecting vaginitis related pathogens | |
| CN117344044A (en) | Composition, kit and application for detecting pathogen related to lung infection | |
| CN117327812A (en) | Composition, kit and application for detecting vaginitis related pathogens | |
| CN109207623B (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and detection kit for identifying three sporothrix bacteria | |
| CN116904621A (en) | Compositions, kits, methods and uses for detecting different genital tract pathogens | |
| CN117802253A (en) | Composition, kit and application for detecting infection-related pathogens | |
| CN117467791A (en) | Primer pair, method and kit for detecting cryptococcus neoformans | |
| CN116004869A (en) | Composition for detecting staphylococcus haemolyticus, enterobacter cloacae and streptococcus pneumoniae | |
| CN117431331A (en) | Composition, kit and application for detecting bacterial respiratory pathogens | |
| CN117802254A (en) | Composition, kit and application for detecting septicemia related pathogens | |
| CN114480689A (en) | RPA method for detecting streptococcus pneumoniae, special primer and probe thereof and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |