Composition, kit and method for detecting blood flow infection related pathogens and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of blood flow infection related pathogens, and more particularly relates to detection and distinguishing of at least four pathogens in burkholderia cepacia, streptococcus mirabilis, staphylococcus capitis, staphylococcus coriolis, proteus mirabilis and staphylococcus haemolyticus. According to the combined detection composition provided by the invention, different pathogens are detected by mainly utilizing a multiple fluorescent PCR analysis method and detecting target spots on the different pathogens; therefore, at least four pathogens in burkholderia cepacia, streptococcus mitis, staphylococcus capitis, staphylococcus coriolis, proteus mirabilis and staphylococcus haemolyticus are detected and distinguished in a single-tube reaction system at the same time, and the detection efficiency is improved. The composition disclosed by the invention is high in detection sensitivity, and the detection sensitivity reaches 200 copies / mL; the reagent / kit provided by the invention adopts a UNG enzyme and dUTP anti-pollution system, so that the accuracy of the detection effect can be guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological detection, in particular, to the detection of pathogens related to bloodstream infection, and more particularly, to the detection and identification of Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus. Background Art

[0002] Burkholderia cepacia is one of several important conditional pathogens of nosocomial infection. It is usually not pathogenic to healthy humans, but often invades individuals with low immunity. It often colonizes the lungs of patients with cystic fibrosis and chronic granulomatous diseases. It is a highly toxic and lethal pathogen. In recent years, the clinical isolation rate of this bacterium has continued to increase, and it has high resistance to multiple antibiotics, which makes 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. It is one of the common pathogens of nosocomial infection and can be isolated from hospital tap water, thermometers, sprayers, catheters, etc. It can also be detected from various clinical samples such as alveolar lavage fluid, sputum, blood, puncture tissue, pus, etc. 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. 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.

[0003] The mitis group includes Streptococcus mitis (S.mitis), oral Streptococcus, infant Streptococcus, pan-oral Streptococcus, and ridge Streptococcus. Streptococcus mitis is a type of viridans streptococcus and is one of the normal flora of the human body. It is mainly distributed in the oropharynx and can also be found in the skin, gastrointestinal tract, and female reproductive tract. Streptococcus mitis is a Gram-positive coccus that is spherical or elliptical. It forms short or long chains in serum broth. It is non-spore, non-motile, facultatively anaerobic, and catalase-negative. Streptococcus mitis is a conditional pathogen that can cause a series of invasive diseases, especially in patients with immunodeficiencies such as malignant tumors, chemotherapy, and neutropenia. Streptococcus mitis can cause bloodstream infections, infective endocarditis, meningitis, pneumonia, odontogenic infections, sinusitis, etc., and can also cause abscess lesions. Streptococcus mitis has the same gene structure encoding gentamicin resistance as Enterococcus faecalis and Enterococcus faecium. This gene determinant cluster is integrated into the chromosome rather than the plasmid. Infection with Streptococcus mitis is not uncommon. There are many causes and clinical types of infection, but it is often missed because it is misidentified as viridans streptococcus in culture. However, the drug resistance of Streptococcus mitis develops faster than that of viridans streptococcus. Therefore, microbiology laboratories and clinicians should strengthen their vigilance against infection with this bacterium and improve the level of diagnosis.

[0004] Staphylococcus capitis is a common coagulase-negative staphylococcus, named because it is often isolated from the human head, face, and neck. Staphylococcus capitis is a Gram-positive bacterium with a cocci-shaped microscopic shape and arranged in a V or W shape. Staphylococcus capitis is frequently detected in sputum and secretions, and the main types of disease are lung infection and wound infection. As a conditional pathogen, this bacterium can cause various serious infections, such as mastitis, complicated skin and soft tissue infection, artificial joint infection, endocarditis, pacemaker-related infection, and neonatal hospital-acquired delayed sepsis. Staphylococcus capitis has more than 3 types of drug resistance and is a multidrug-resistant bacterium, which should be taken seriously in clinical practice. Most patients with bloodstream infection have low immunity. The genus of Staphylococcus capitis, which is a conditional pathogen, may become a pathogen in this population, so it cannot be ignored in clinical treatment.

[0005] Staphylococcus cohnii belongs to the coagulase-negative staphylococcus, a skin commensal bacteria, which is often distributed in the environment of humans, livestock and food. This bacterium is one of the normal strains that settle on human skin and mucous membranes, and its pathogenicity is very low under normal circumstances. In recent years, with the increase in invasive procedures for indwelling intravenous catheters, Staphylococcus cohnii has become an important pathogen. Another way for humans to be infected with Staphylococcus cohnii is milk. The microbial contamination in milk mainly comes from the cow's body and from the outside during milking, storage and transportation. There are more and more reports of infections caused by Staphylococcus cohnii, which can cause harm to human and animal health and the breeding industry, so it has attracted attention.

[0006] Proteus mirabilis belongs to the genus Proteus of the family Morganellaceae. It is a Gram-negative, facultatively anaerobic, blunt-ended rod. Proteus mirabilis is an important conditional pathogen, widely distributed in the natural environment and the intestines of humans and animals. At present, Proteus mirabilis has become one of the main pathogens of nosocomial infections and is closely related to urinary tract infections. Proteus mirabilis and Proteus vulgaris are the main pathogens of urinary tract infections after Escherichia coli. The formation of kidney stones and bladder stones may be related to Proteus infection. Some strains can cause sepsis, food poisoning, peritonitis, meningitis, etc.

[0007] Staphylococcus haemolyticus is a coagulase-negative staphylococcus, which belongs to the normal flora of human skin and mucous membranes. When the body's flora is disturbed, it can cause suppurative infections in the blood, urogenital tract, and trauma. The isolation rate of Staphylococcus haemolyticus among staphylococci has increased significantly, especially in bloodstream infections. Staphylococcus haemolyticus is the second most commonly isolated coagulase-negative staphylococcus from clinical cases. Staphylococcus haemolyticus has strong adaptability and survival ability in the hospital environment, especially on medical devices, and has become an important factor in nosocomial infections of multidrug-resistant staphylococci. In recent years, with the widespread use of broad-spectrum antibiotics, the drug resistance of Staphylococcus haemolyticus has become increasingly serious. Early diagnosis of pathogens has guiding significance for the use of antibiotics.

[0008] The above-mentioned Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis, and Staphylococcus haemolyticus can all cause sepsis and endanger life safety. 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. However, it is difficult to identify the type of infected bacteria through clinical symptoms and routine laboratory tests, and the current bacterial culture conditions are relatively harsh, resulting in a low culture positive rate, and the culture of multiple mixed infections is extremely difficult, which brings great trouble to infected patients and clinicians.

[0009] Currently, the commonly used single-tube multiplex detection systems mostly use the melting curve method. By selecting different fluorescent signals, dozens of pathogens can be detected in a single tube. However, the sensitivity of this technology is low and it is difficult to reach less than 3000 copies / mL.

[0010] Therefore, there is an urgent need in this field for a product that can simply and quickly detect and identify the above-mentioned multiple pathogens, so as to identify the pathogen, diagnose early, and reasonably guide antibiotic treatment. Summary of the invention

[0011] The existing detection methods are complex, with high missed detection rate, slow reaction speed and low detection precision. In view of this, in a first aspect, the present invention provides a composition for detecting pathogens related to bloodstream infection, including primer pairs and probes for detecting at least four pathogens among Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus:

[0012] Primer pairs and probes for detecting Burkholderia cepacia as shown in SEQ ID NOs: 1 to 3;

[0013] Primer pairs and probes for detecting Streptococcus mitis as shown in SEQ ID NOs: 4 to 6;

[0014] A primer pair and a probe for detecting Staphylococcus capitis as shown in SEQ ID NOs: 7 to 9;

[0015] A primer pair and a probe for detecting Staphylococcus cohnii as shown in SEQ ID NOs: 10 to 12;

[0016] A primer pair and a probe for detecting Proteus mirabilis as shown in SEQ ID NOs: 13 to 15; and

[0017] The primer pair and probe for detecting hemolytic Staphylococcus are shown in SEQ ID NOs: 16-18.

[0018] The present invention provides a combined detection composition, which mainly uses a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and identification of at least four pathogens among Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus in a single tube reaction system. The composition of the present invention has a higher detection sensitivity of 200 copies / mL, good specificity, and more accurate detection.

[0019] The most common method for pathogen detection is fluorescent quantitative PCR (polymerase chain reaction). The PCR reaction system contains a pair of specific primers and a Taqman probe. The probe is a specific oligonucleotide sequence with a reporter fluorescent group and a quencher fluorescent group labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; if there is a target sequence in the reaction system, the probe binds to the template during the PCR reaction, and the DNA polymerase uses the exonuclease activity of the enzyme to enzymatically degrade the probe along the template, and the reporter group separates from the quencher group and emits fluorescence. For each amplified DNA chain, a fluorescent molecule is generated. The fluorescent quantitative PCR instrument can monitor the number of cycles (Ct value) at which the fluorescence reaches a preset threshold value, which is related to the concentration of pathogen nucleic acid. The higher the concentration of pathogen nucleic acid, the smaller the Ct value. In the end, the infection can be determined based on the Ct value.

[0020] In some embodiments, the composition of the present invention may include one or more sets of the above primer pairs and probes. In this article, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0021] Furthermore, the composition of the present invention can be arbitrarily combined into any combination of detection corresponding to 6 targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe sets corresponding to the targets. These combinations are all included in the present invention.

[0022] For example, it may include all combinations of the above-mentioned 6 groups of primer pairs and probes, may include any 5 groups of the above-mentioned 6 groups of primer pairs and probes, may include any 4 groups of the above-mentioned 6 groups of primer pairs and probes, may include any 3 groups of the above-mentioned 6 groups of primer pairs and probes, may include any 2 groups of the above-mentioned 6 groups of primer pairs and probes, and may also include any 1 group of the above-mentioned 6 groups of primer pairs and probes.

[0023] Furthermore, the fluorescent groups of the composite probes are different from each other and do not interfere with each other.

[0024] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO425, FAM, HEX, ROX, CY5 and Quasar 705 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.

[0025] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0026] In some specific embodiments, the probes in the compositions of the present invention are labeled with a fluorescent reporter group at the 5' end.

[0027] Further, the fluorescent reporter group of the probe for detecting Burkholderia cepacia as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting Streptococcus mitis as shown in SEQ ID NO:6 is HEX; the fluorescent reporter group of the probe for detecting Staphylococcus capitis as shown in SEQ ID NO:9 is ROX; the fluorescent reporter group of the probe for detecting Staphylococcus cohnii as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting Proteus mirabilis as shown in SEQ ID NO:15 is Quasar 705; and the fluorescent reporter group of the probe for detecting Staphylococcus hemolyticus as shown in SEQ ID NO:18 is ATTO 425.

[0028] In some specific embodiments, the 3' end of the probe further has a fluorescence quenching group, such as MGB, BHQ1 or BHQ2.

[0029] Furthermore, the components of the composition exist in a mixed form.

[0030] In a second aspect, provided is the use of the composition of the present invention in preparing a kit for the joint detection and differentiation of bloodstream infection-related pathogens, wherein the pathogens are Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus.

[0031] In a third aspect, the present invention provides a kit for the joint detection and differentiation of pathogens associated with bloodstream infection, the kit comprising the composition of the present invention.

[0032] Furthermore, in each reaction system, the amount of a single primer is 0.01-0.5 μM, and the amount of a single probe is 0.005-0.2 μM.

[0033] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0034] In a specific embodiment, the negative control is physiological saline, but is not limited to physiological saline, and can also be at least one of DEPC H2O and internal standard gene.

[0035] Commonly used internal standards include exogenous non-human genome internal standards, human genome internal standards, artificially synthesized internal standards, etc.

[0036] In a specific embodiment, the positive control is at least one of a fragmented plasmid or a fragmented DNA of Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis, and Staphylococcus haemolyticus.

[0037] Furthermore, the kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a UNG enzyme, a DNA polymerase, dNTPs (U) and Mg 2+ At least one of .

[0038] In some specific embodiments, the DNA polymerase is Taq enzyme.

[0039] In a specific embodiment, the kit of the present invention comprises PCR buffer, UNG enzyme, Taq enzyme, dNTPs (U), primers, probe and Mg 2+ .

[0040] Common PCR buffers are composed of Tris-HCl, KCl, MgCl2 and other buffer systems. Generally, the total volume in a single PCR reaction tube is 50 μL.

[0041] In a fourth aspect, the present invention provides a method for joint detection and differentiation of bloodstream infection-related pathogens for non-diagnostic purposes, the method comprising the following steps:

[0042] (1) Extracting or releasing nucleic acid from the sample to be tested;

[0043] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0044] (3) Obtain and analyze the results.

[0045] In the present invention, the sample used for detection is blood, but is not limited thereto.

[0046] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0047] UNG enzyme reaction, temperature is 50℃, time is 5min, 1 cycle; Taq enzyme activation, temperature is 95℃, time is 1min; denaturation, temperature is 95℃, time is 10s, annealing, extension and fluorescence collection, temperature is 60℃, time is 20s, 41 cycles.

[0048] Furthermore, the reaction conditions of the fluorescent quantitative PCR also include: instrument cooling, temperature of 25° C., time of 10 s, and 1 cycle.

[0049] In a specific embodiment, the present invention provides a composition for preparing a reagent for joint detection and differentiation of bloodstream infection-related pathogens, the use comprising the following steps:

[0050] (1) Extracting or releasing nucleic acid from the sample to be tested;

[0051] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0052] (3) Obtain and analyze the results.

[0053] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0054] UNG enzyme reaction, temperature is 50℃, time is 5min, 1 cycle; Taq enzyme activation, temperature is 95℃, time is 1min; denaturation, temperature is 95℃, time is 10s, annealing, extension and fluorescence collection, temperature is 60℃, time is 20s, 41 cycles.

[0055] Furthermore, the reaction conditions of the fluorescent quantitative PCR also include: instrument cooling, temperature of 25° C., time of 10 s, and 1 cycle.

[0056] Herein, the term "non-diagnostic purpose" means not intended to obtain information on whether an individual is infected with the above pathogens and suffers from diseases such as urinary tract infection. For example, the presence of the above pathogens can be detected in a test culture (such as urine, etc.).

[0057] In the present invention, the term "primer pair" refers to two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target gene, and the other primer is complementary to another DNA template strand at the other end of the target gene. Alternatively, it refers to two oligonucleotide chains used to specifically amplify the target DNA fragment in the polymerase chain reaction (PCR). This pair of primers corresponds to the two ends of the target DNA sequence, one is complementary to the 5' end of the template strand, called the upstream primer or forward primer; the other is complementary to the 3' end of the template strand, called the downstream primer or reverse primer.

[0058] In the present invention, the term "probe" refers to a nucleic acid sequence (DNA or RNA) with a detection label, a known sequence, and complementary to the target gene.

[0059] In the present invention, the term "nucleotide sequence" refers to the order of bases in DNA or RNA.

[0060] The above scheme of the present invention has the following beneficial effects:

[0061] (1) The present invention is used to detect and differentiate pathogens related to bloodstream infection, including the detection and differentiation of at least four pathogens such as Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus. The combined detection composition provided by the present invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and differentiation of at least four pathogens such as Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus in a single tube reaction system, thereby improving the detection efficiency. The composition of the present invention has a higher detection sensitivity of 200 copies / mL; the reagent / kit of the present invention adopts UNG enzyme and dUTP anti-pollution system, which can ensure the accuracy of the detection effect.

[0062] (2) The present invention optimizes the PCR reaction conditions and establishes a multiplex real-time fluorescence quantitative RTPCR reaction system. In particular, for the low load characteristics of blood-borne samples, PCR fluorescence detection can avoid the phenomenon of false negatives compared to melting curves, and has higher sensitivity. It can also detect Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis, and Staphylococcus haemolyticus at the same time. The detection results of the present invention have the advantages of being sensitive and rapid, highly specific, less contaminating, and easy to standardize, and can be widely used in laboratory testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0064] Figure 1 It is an amplification curve diagram of the combined detection of Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus by the composition of the present invention;

[0065] Figure 2 It is an amplification curve diagram of the combined detection of Burkholderia cepacia, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus by the composition of the present invention;

[0066] Figure 3 It is an amplification curve diagram of the combined detection of Burkholderia cepacia, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus by the composition of the present invention;

[0067] Figure 4is a sensitivity result graph of the composition of the present invention (Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus);

[0068] Figure 5 is a blank-specific result graph of the composition of the present invention;

[0069] Figure 6 It is an amplification curve diagram of a single hemolytic Staphylococcus test in an embodiment of the present invention.

[0070] Figure 7 It is an amplification curve diagram of a single hemolytic Staphylococcus test in the comparative example of the present invention.

[0071] Figure 8 It is an amplification curve diagram of the comparative example composition of the present invention for joint detection of Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus. DETAILED DESCRIPTION

[0072] The present invention aims at the existing problems and provides a composition, a kit, a method and its use for detecting pathogens related to bloodstream infection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0073] The disclosed embodiments are described to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

[0074] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0075] Example 1: Primers and probes used in the present invention

[0076] The primer pairs and probes used in the present invention are shown in Table 1 below.

[0077] Table 1

[0078]

[0079] Among them, the fluorescent reporter group of the probe for detecting Burkholderia cepacia as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting Streptococcus mitis as shown in SEQ ID NO:6 is HEX; the fluorescent reporter group of the probe for detecting Staphylococcus capitis as shown in SEQ ID NO:9 is ROX; the fluorescent reporter group of the probe for detecting Staphylococcus cohnii as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting Proteus mirabilis as shown in SEQ ID NO:15 is Quasar 705; and the fluorescent reporter group of the probe for detecting Staphylococcus hemolyticus as shown in SEQ ID NO:18 is ATTO 425.

[0080] Example 2: Method for detecting pathogens

[0081] (1) Reagent preparation:

[0082] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture according to the following ratio, mix them thoroughly to form a PCR mixture, and set aside.

[0083] Physiological saline was used as a negative control, and a mixed plasmid containing the specific gene target sequences listed in Table 1 was used as a positive control.

[0084] Preparation of enzyme mixture:

[0085] The enzyme mixture consists of UNG enzyme and Taq enzyme, which are mixed at a ratio of 9:1.

[0086] (2) Sample processing and sample addition

[0087] Place the test samples, negative controls, and positive controls into 1.5 mL centrifuge tubes respectively, and use conventional nucleic acid extraction or purification reagents according to their instructions to extract nucleic acids.

[0088] The treated samples, negative controls and positive controls were pipetted into reaction tubes respectively, and 50 μL of PCR mixture was added to each reaction tube and the tube cap was covered.

[0089] The real-time fluorescence PCR reaction system is configured according to Table 2 below:

[0090] Table 2

[0091] Components Volume / concentration in each reaction PCR buffer 25.09μL Enzyme mixture 2μL (5U / μL) dNTPs(U) 1.6μL <![CDATA[Mg 2+ ]]> 0.34μL(1M) Primer (50pmol / uL) 0.33μL / strip Probe (50pmol / uL) 0.15μL / strip Sample DNA 10μL <![CDATA[ddH2O]]> Make up to 50 μL

[0092] Note: 0.33 μL / strip means that the volume of each primer is 0.33 μL, and the present invention has 12 primers in total; 0.15 μL / strip means that the volume of each probe is 0.15 μL, and the present invention has 6 probes in total.

[0093] (3) The amplification reaction procedure is shown in Table 3.

[0094] Table 3

[0095]

[0096] (4) Testing, result analysis and judgment

[0097] After the reaction is completed, the results are automatically saved, and the amplification curves of the detection targets are analyzed separately. According to the analyzed image, 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 physiological saline 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.

[0098] Quality Control

[0099] Negative control: No Ct value is displayed in the curves of the six channels of ATTO 425, FAM, HEX, ROX, CY5, and Quasar 705;

[0100] Positive control: Ct≤36 for the six channels of ATTO 425, FAM, HEX, ROX, CY5, and Quasar 705;

[0101] The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be invalid and must be repeated.

[0102] Positive judgment value

[0103] Through the above reference value research, it was determined that the Ct reference value of this reagent for detecting the target gene is 38.

[0104] Interpretation of Test Results

[0105] If the ATTO 425, FAM, HEX, ROX, CY5 and Quasar 705 channels have obvious S-shaped amplification curves and Ct values ​​≤ 38, they are judged as positive; if the ATTO 425, FAM, HEX, ROX, CY5 and Quasar 705 channels have no amplification curve (No Ct) or Ct values ​​> 38, they are judged as negative. The details are shown in Table 4:

[0106] Table 4

[0107]

[0108] Example 3: Test results of the test samples of the composition of the present invention

[0109] The primer pairs and probes shown in Example 1 were verified on samples of Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus according to the method of Example 2. The results showed that Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus could be jointly detected and distinguished.

[0110] Among them, the results of the joint test for Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus were as follows: Figure 1 shown.

[0111] The results of the joint test for Burkholderia cepacia, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus are as follows Figure 2 shown.

[0112] The results of the joint test for Burkholderia cepacia, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus are as follows Figure 3 shown.

[0113] from Figures 1 to 3 It can be seen that the composition of the present invention can perform joint detection and differentiation on four, five or six of the above pathogens.

[0114] Example 4: Sensitivity of the composition of the present invention

[0115] The composition in Example 1 of the present invention was used to perform LOD (sensitivity) detection on each pathogen to simulate clinical samples, and multiple PCR detection was performed on a Macrostone fluorescent quantitative PCR instrument. The detection rate results are shown in Table 5, and the sensitivity results are shown in Table 5. Figure 4 As shown, the test results show that the composition of the present invention can still accurately detect each channel of samples as low as 200 copies / mL, indicating that the sensitivity of the composition of the present invention is 200 copies / mL.

[0116] Table 5

[0117]

[0118]

[0119] Example 5. Specificity of the composition of the present invention

[0120] Using the fluorescent PCR reaction system in Example 2 of the present invention, with physiological saline as the sample, a blank specificity test was performed on the composition for detecting bloodstream infection-related pathogens in Example 1. The test results are as follows: Figure 5 As shown, there is no non-specific amplification in each target channel, and the blank specificity of the kit of the present invention is good.

[0121] Comparative Example 1: Other primers and probes designed by the present invention with poor effects

[0122] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be excluded at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. It is necessary to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection) and consider the mutual interference between different primers and probes, so the primers and probes need to be carefully designed. Therefore, the inventors designed some other primers and probes.

[0123] This comparative example refers to Example 2 to detect bloodstream infection-related pathogens. The difference between this comparative example and Example 2 is that the primer pair probe sequence of hemolytic Staphylococcus is different. In addition to the above differences, the other materials and process operations used in this comparative example are strictly consistent with those in Example 2. The primer pair probe sequence of hemolytic Staphylococcus in this comparative example is shown in Table 6 below.

[0124] The primer pair and probe (SEQ ID NO: 16-18) in Example 1 and the primer pair and probe (SEQ ID NO: 19-21) in Comparative Example 1 were respectively used to perform single detection of hemolytic Staphylococcus aureus, and the detection results were as follows: Figure 6 and 7 As shown, hemolytic Staphylococcus aureus-1 is from Example 1, and hemolytic Staphylococcus aureus-2 is from Comparative Example 1.

[0125] The primer pair and probe (SEQ ID NO: 19-21) in Comparative Example 1 were used to replace the primer pair and probe (SEQ ID NO: 16-18) in Example 1, and the primer pair and probe (SEQ ID NO: 1-15) in Example 1 were combined to form a composition (SEQ ID NO: 1-15 and SEQ ID NO: 19-21) to jointly detect the specific genes of Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that the detection of the composition for detecting bloodstream infection-related pathogens in Comparative Example 1 (primer pair probe sequences SEQ ID NOs: 1 to 15 and SEQ ID NOs: 19 to 21) in the six-in-one detection system was affected, and the detection performance of hemolytic Staphylococcus in its amplification curve was reduced, the Ct value was delayed, and the fluorescence increment decreased.

[0126] from Figures 6 to 8It can be seen that the effects of the two single tests are not much different, but the effect becomes worse after the joint test. This fully demonstrates the superiority of the composition of the present invention, and the mutual interference between different primer pairs and probes is small. In other words, the combination of primers and probes in the present invention is irreplaceable and substitutable.

[0127] Table 6

[0128]

[0129] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for joint detection and differentiation of pathogens related to bloodstream infection, characterized in that: Includes primer pairs and probes for detection of at least 4 pathogens among Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis, and Staphylococcus haemolyticus: Primer pairs and probes for detecting Burkholderia cepacia as shown in SEQ ID NOs: 1 to 3; Primer pairs and probes for detecting Streptococcus mitis as shown in SEQ ID NOs: 4 to 6; A primer pair and a probe for detecting Staphylococcus capitis as shown in SEQ ID NOs: 7 to 9; A primer pair and a probe for detecting Staphylococcus cohnii as shown in SEQ ID NOs: 10 to 12; A primer pair and a probe for detecting Proteus mirabilis as shown in SEQ ID NOs: 13 to 15; and The primer pair and probe for detecting hemolytic Staphylococcus are shown in SEQ ID NOs: 16-18.

2. The composition according to claim 1, characterized in that The fluorescent groups of the composite probes are different from each other and do not interfere with each other.

3. The composition according to claim 1, characterized in that The fluorescent reporter group of the probe for detecting Burkholderia cepacia as shown in SEQ ID NO:3 is FAM; the fluorescent reporter group of the probe for detecting Streptococcus mitis as shown in SEQ ID NO:6 is HEX; the fluorescent reporter group of the probe for detecting Staphylococcus capitis as shown in SEQ ID NO:9 is ROX; the fluorescent reporter group of the probe for detecting Staphylococcus cohnii as shown in SEQ ID NO:12 is CY5; the fluorescent reporter group of the probe for detecting Proteus mirabilis as shown in SEQ ID NO:15 is Quasar 705; the fluorescent reporter group of the probe for detecting Staphylococcus haemolyticus as shown in SEQ ID NO:18 is ATTO 425.

4. The composition according to any one of claims 1 to 3, characterized in that The components of the composition are present in admixture.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a reagent or kit for joint detection and differentiation of pathogens related to bloodstream infection, wherein: The pathogens are Burkholderia cepacia, Streptococcus mitis, Staphylococcus capitis, Staphylococcus cohnii, Proteus mirabilis and Staphylococcus haemolyticus.

6. A kit for joint detection and differentiation of pathogens related to bloodstream infection, characterized in that: The kit comprises the composition according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that In each reaction system, the amount of a single primer is 0.01-0.5 μM, and the amount of a single probe is 0.005-0.2 μM.

8. The kit according to claim 6 or 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to any one of claims 6 to 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a UNG enzyme, a DNA polymerase, dNTPs (U) and Mg 2+ At least one of .

10. A method for joint detection and differentiation of pathogens associated with bloodstream infection for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) extracting or releasing nucleic acid from the sample to be tested; (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition according to any one of claims 1 to 4 or the kit according to any one of claims 6 to 9; (3) Obtain and analyze the results.

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