Composition for detecting blood flow infection related pathogens

Abstract

The invention belongs to the field of molecular biological detection, and provides a joint detection composition which is mainly used for detecting related pathogens by using a multiple fluorescent PCR (Polymerase Chain Reaction) analysis method. Therefore, at least four of the klebsiella aerogenes, the stenotrophomonas maltophilia, the streptococcus angina, the acinetobacter baumannii, the klebsiella pneumoniae and the klebsiella oxytoca can be simultaneously detected and distinguished in a single-tube reaction system. Meanwhile, the detection sensitivity of the composition is higher and reaches 500 copies / mL, and the detection is more accurate.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to a composition for detecting and distinguishing pathogens related to bloodstream infection, including Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, and Klebsiella oxytoca. Background Art

[0002] Klebsiella aerogenes is a species of Klebsiella, formerly known as Enterobacter aerogenes. It is a Gram-negative bacillus, oxidase-negative, catalase-positive, citric acid test-positive, and indole test-negative. It can ferment a variety of sugars and produce a large amount of gas. Klebsiella aerogenes exists in the intestines of humans and animals, as well as in water, soil, and decayed matter, and is an opportunistic pathogen. Under certain conditions, it can cause urinary tract infections, respiratory tract infections, wound infections, and sepsis in humans. It can also cause hospital infections, such as urinary tract infections after catheterization. Most strains are sensitive to antibiotics, but are prone to develop drug resistance during treatment.

[0003] Stenotrophomonas maltophilia belongs to the Xanthomonadaceae family of the order Xanthomonadales. It is a Gram-negative conditional pathogen that is widely present in nature and hospital environments. With the widespread use of broad-spectrum antibiotics and immunosuppressants, as well as the increasing number of invasive procedures, its isolation rate has been on the rise among non-fermenting bacteria. Due to its weak pathogenicity, its infection often occurs in patients with low immunity and critical conditions, usually hospital infections, and rarely occurs in the community. In addition, the bacteria are inherently resistant to a variety of antimicrobial drugs, posing challenges to clinical anti-infection treatment and infection prevention and control. The most common type of infection is lower respiratory tract infection, which can also cause infections in the bloodstream, urinary system, abdominal cavity, skin and soft tissue. In addition, Stenotrophomonas maltophilia often forms mixed infections with other conditional pathogens. Infections caused by Stenotrophomonas maltophilia can occur in children and adults, especially in immunocompromised patients. Risk factors for infection include: malignant tumors, indwelling central venous catheters, chronic respiratory diseases, low immune function, hypoproteinemia, long-term treatment with broad-spectrum antibiotics, and long hospital stays (especially in the ICU). Direct contact is the main mode of transmission, and the hands of medical staff are the most common medium of transmission, which can infect susceptible people through contact between people and between people and surfaces.

[0004] Streptococcus anginosus is one of the normal flora colonized in the oral cavity, throat, digestive tract, etc. It is one of the Miller Streptococcus group, including Streptococcus anginosus, Streptococcus intermedius and Streptococcus constellatus. After scholars discovered Streptococcus anginosus in 1906, scholars have continuously reported the isolation of this bacterium from infected specimens of patients. The most common diseases caused by Streptococcus anginosus are respiratory tract infections, suppurative infections and bloodstream infections, accounting for 34.8%, 33.5% and 8.5% respectively. Previous studies have confirmed that Streptococcus anginosus can produce a variety of toxins in vitro, which often cause invasive suppurative infections. Once it invades the blood, it can spread throughout the body with the blood circulation, causing severe systemic infectious diseases. Most patients with bloodstream infections caused by Streptococcus anginosus are related to invasive operations. The clinical characteristics of bloodstream infections caused by Streptococcus anginosus are that most patients have some underlying diseases, such as digestive system, cardiovascular system, respiratory system diseases and tumors. The characteristics of streptococcal infection in pharyngitis are not obvious and identification is difficult. In clinical work, if a related bacterial infection is suspected, specimens should be sent for examination in a timely manner, antibiotics should be used reasonably according to the drug sensitivity results, and drug resistance monitoring should be carried out to improve prognosis.

[0005] 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 bacteria are widely distributed in hospital environments and can survive for a long time, which can easily cause infection in critically ill patients. Therefore, they are often isolated from specimens such as blood, urine, pus and respiratory secretions of infected patients. Among non-fermenting bacteria, infection is second only to Pseudomonas. The infection of Acinetobacter baumannii is gradually increasing, and its drug resistance is becoming increasingly 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 hospital environments 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. The carbapenem-resistant Acinetobacter baumannii is developing rapidly in China, and "pan-drug resistant" Acinetobacter baumannii has recently appeared, which should cause high vigilance.

[0006] Klebsiella pneumoniae is a common Gram-negative bacillus in clinical practice. It is widely distributed in nature and also exists in the respiratory tract and intestines of healthy humans and animals. It can cause infection when the body's immunity is reduced or long-term use of antibiotics leads to dysbiosis. It can cause community-acquired and hospital-acquired infections, leading to pneumonia, liver abscess, urinary tract infection, bloodstream infection and other infectious diseases. In recent years, Klebsiella pneumoniae infection incidents have been high, and it is a potential pathogen that cannot be ignored for causing community-acquired and hospital-acquired infections.

[0007] Klebsiella oxytoca is a Gram-negative coccobacillus with capsule, pili, no flagella, facultative anaerobe, a species of Klebsiella in the family Enterobacteriaceae, and an important conditional pathogen. The bacterium can colonize in the gastrointestinal tract, skin, respiratory tract, nasopharynx, soil, water and other surrounding environments. Some strains can produce heat-resistant enterotoxins, and are important nosocomial and community infection pathogens. Klebsiella oxytoca can cause infections of the respiratory, bloodstream, abdominal cavity and central nervous system of the human body. Studies have shown that Klebsiella oxytoca is an important pathogen of antibiotic-associated hemorrhagic enteritis in children. Klebsiella oxytoca has strong virulence. After infection with the bacterium in the case of imperfect or low immune system function, the bacteria can enter the blood circulation through different pathways and multiply in large numbers. The toxins released cause systemic infection and cause sepsis, thereby leading to multiple organ dysfunction and failure, and even death. Studies have shown that Klebsiella oxytoca infection has obvious seasonal differences, with summer and autumn as the peak incidence period. In the process of clinical treatment, in order to deal with the bacterial infection, the large-scale irregular use of antimicrobial drugs has led to the emergence and spread of multidrug-resistant bacteria, especially the emergence of resistant bacteria producing extended-spectrum β-lactamases, which has brought severe challenges to clinical treatment. In recent years, the emergence and widespread prevalence of carbapenem-resistant bacteria have been particularly prominent. Several studies have shown that carbapenem-resistant Klebsiella oxytoca has been reported in many parts of the world in sporadic or clustered epidemics.

[0008] The above-mentioned Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, and Klebsiella oxytoca 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] Therefore, there is a need in the art for a product that can simply and quickly detect the above-mentioned pathogens with high sensitivity and good specificity. In particular, a product that can directly detect the above-mentioned pathogens in the blood is beneficial for providing timely clinical treatment strategies. Summary of the invention

[0010] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens related to bloodstream infection, comprising upstream and downstream primers and probes for detecting at least 4 of the following pathogens:

[0011] Upstream and downstream primers and probes for detecting Klebsiella aerogenes as shown in SEQ ID NOs: 1 to 3;

[0012] Upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia as shown in SEQ ID NOs: 4 to 6;

[0013] Upstream and downstream primers and probes for detecting Streptococcus anginosus as shown in SEQ ID NOs: 7 to 9;

[0014] Upstream and downstream primers and probes for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 10 to 12;

[0015] Upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 13 to 15; or

[0016] The upstream and downstream primers and probes for detecting Klebsiella oxytoca are shown in SEQ ID NOs: 16-18.

[0017] The joint detection composition provided by the present invention mainly uses a multiplex fluorescent PCR analysis method to detect relevant pathogens, thereby simultaneously realizing the detection and differentiation of at least 4 pathogens among Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, and Klebsiella oxytoca in a single-tube reaction system, so as to provide a targeted strategy for subsequent treatment. At the same time, the composition of the present invention has a higher detection sensitivity of 500 copies / mL, and its detection is more accurate than the melting curve, which is conducive to providing prevention policies in a timely manner.

[0018] The present invention provides a composition for detecting pathogens related to bloodstream infection, comprising upstream and downstream primers and probes for detecting at least five of the above pathogens.

[0019] The present invention provides a composition for detecting pathogens related to bloodstream infection, comprising:

[0020] Upstream and downstream primers and probes for detecting Klebsiella aerogenes as shown in SEQ ID NOs: 1 to 3;

[0021] Upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia as shown in SEQ ID NOs: 4 to 6;

[0022] Upstream and downstream primers and probes for detecting Streptococcus anginosus as shown in SEQ ID NOs: 7 to 9;

[0023] Upstream and downstream primers and probes for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 10 to 12;

[0024] Upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 13 to 15; and

[0025] The upstream and downstream primers and probes for detecting Klebsiella oxytoca are shown in SEQ ID NOs: 16-18.

[0026] Furthermore, the composition includes upstream and downstream primers and probes for detecting the internal standard.

[0027] In some specific embodiments, the internal standard is a human internal standard gene.

[0028] In a specific embodiment, the internal standard is a human housekeeping gene.

[0029] Furthermore, the fluorescent groups of different probes in the composition of the present invention are different from each other and do not interfere with each other.

[0030] 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, Quasar705, FAM, HEX, ROX, CY5 and CY5.5 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.

[0031] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe sets. In the present invention, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0032] The composition of the present invention can be combined into any combination of the corresponding six targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention. The effect of being able to detect the corresponding target combination is also expected by those skilled in the art.

[0033] For example, it may include any 5 of the above 6 sets of primers and probes, it may include any 4 of the above 6 sets of primers and probes, it may include any 3 of the above 6 sets of primers and probes, it may include any 2 of the above 6 sets of primers and probes, or it may include any 1 of the above 6 sets of primers and probes.

[0034] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0035] In a specific embodiment, the fluorescent reporter group of the Klebsiella aerogenes probe is FAM; the fluorescent reporter group of the Stenotrophomonas maltophilia probe is HEX; the fluorescent reporter group of the Streptococcus anginosus probe is ROX; the fluorescent reporter group of the Acinetobacter baumannii probe is CY5; the fluorescent reporter group of the Klebsiella pneumoniae probe is Quasar 705; and the fluorescent reporter group of the Klebsiella oxytoca probe is ATTO425.

[0036] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0037] Furthermore, the 3' end of the probe also has a quencher group, such as MGB, Dabcyl BHQ-0, BHQ1, BHQ2 or BHQ3.

[0038] Furthermore, the dosage of a single primer in the composition is 0.01 to 0.5 μM; the dosage of a single probe in the composition is 0.005 to 0.2 μM.

[0039] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0040] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0041] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0042] In a second aspect, the present invention provides the use of the above-mentioned composition of the present invention in the preparation of a kit for detecting bloodstream infection-related pathogens, wherein the type of the related pathogens is one or more of Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, and Klebsiella oxytoca.

[0043] In a third aspect, the present invention provides a kit for detecting pathogens associated with bloodstream infection, the kit comprising the composition of the present invention as described above.

[0044] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0045] In a specific embodiment, the negative control is at least one of DEPC H2O, physiological saline, and internal standard genes. The positive control is at least one of the plasmid or fragment genes of Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, and Klebsiella oxytoca.

[0046] Furthermore, the kit also includes a nucleic acid amplification reagent.

[0047] Furthermore, the amplification reagent includes dNTP(U)s, PCR buffer, DNA polymerase, and Mg 2+ At least one of .

[0048] Furthermore, the kit also includes: a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0049] Furthermore, the concentration of the DNA polymerase is 1 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0050] In a specific embodiment, the kit of the present invention comprises: Taq enzyme, Mg 2+ , dNTP(U)s, primers, probes, UNG enzyme and PCR buffer.

[0051] PCR buffer is composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume in a single PCR reaction tube is generally 20 μl to 200 μl.

[0052] In a fourth aspect, a method for detecting bloodstream infection-related pathogens is provided, the method comprising the following steps:

[0053] 1) Extracting nucleic acid from the sample to be tested;

[0054] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0055] 3) Obtain and analyze the results.

[0056] In the present invention, the sample used for detection may be serum, blood, etc., but is not limited thereto.

[0057] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0058] UNG enzyme reaction, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0059] In a specific embodiment, a method for detecting bloodstream infection-related pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

[0060] 1) Extracting nucleic acid from the sample to be tested;

[0061] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0062] 3) Obtain and analyze the results.

[0063] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0064] UNG enzyme reaction, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0065] In a specific embodiment, a composition is provided for use in preparing a reagent for detecting pathogens associated with bloodstream infection, wherein the use detects the following steps:

[0066] 1) Extracting nucleic acid from the sample to be tested;

[0067] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent quantitative PCR on the nucleic acid obtained in step 1);

[0068] 3) Obtain and analyze the results.

[0069] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0070] UNG enzyme reaction, temperature is 50-65°C, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99°C, time is 1-5 minutes, 1 cycle; denaturation, temperature is 95°C, time is 5-30 seconds, annealing, temperature is 55°C-60°C, time is 10-60 seconds, 30-50 cycles, and fluorescence is collected.

[0071] In this context, the term "non-diagnostic purpose" means that the method is not intended to obtain information on whether an individual is infected with the above pathogens and suffers from a bloodstream infection. For example, the method can detect whether the environment and culture contain the above pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figures 1 to 3 A graph showing the test results of different sample combinations of the composition of the present invention;

[0073] Figure 4 It is the sensitivity result diagram of the composition of the present invention;

[0074] Figure 5 It is a specific result diagram of the composition of the present invention;

[0075] Figure 6 It is a test result diagram of the comparative example composition of the present invention. DETAILED DESCRIPTION

[0076] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0077] Example 1: Primers and probes used in the present invention

[0078] Table 1

[0079]

[0080]

[0081] Among them, the fluorescent reporter group of the Klebsiella aerogenes probe is FAM; the fluorescent reporter group of the Stenotrophomonas maltophilia probe is HEX; the fluorescent reporter group of the Streptococcus anginosus probe is ROX; the fluorescent reporter group of the Acinetobacter baumannii probe is CY5; the fluorescent reporter group of the Klebsiella pneumoniae probe is Quasar 705; and the fluorescent reporter group of the Klebsiella oxytoca probe is ATTO425.

[0082] Example 2: Method for detecting bloodstream infection-related pathogens

[0083] The test sample of the present invention is blood. The real-time fluorescence PCR reaction solution is configured according to the following Table 2:

[0084] Table 2

[0085] Components Volume / concentration in each reaction PCR Buffer 25.09μL Enzyme Mix 2μL (5U / μL) <![CDATA[Mg 2+ ]]> 0.34μL(1M) dNTPs(U) 1.6μL Primer probe 0.33μL / 0.15μL Sample DNA 10μL <![CDATA[ddH2O]]> Make up to 50 μL

[0086] Sample handling and loading:

[0087] Take 300 μL of the sample to be tested, negative control, and positive control into a 1.5 mL centrifuge tube, and use the nucleic acid extraction or purification reagent of Shengxiang Biotechnology Co., Ltd. according to its instructions to extract nucleic acid.

[0088] 10 μL of the above-treated samples, negative controls and positive controls were pipetted and added to reaction tubes respectively. 40 μL of PCR mixture was added to each reaction tube and the tube cap was covered.

[0089] PCR amplification:

[0090] On the fully automatic medical PCR analysis system, PCR amplification is performed according to a certain temperature and time setting program. The PCR amplification program is set as shown in Table 3 below:

[0091] Table 3

[0092]

[0093] 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 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.

[0094] Quality Control

[0095] Negative control: no Ct value is displayed in the curves of the three channels of ATTO425, FAM, HEX, ROX, CY5, and Quasar 705;

[0096] Positive control: Ct≤36 for the three channels of ATTO425, FAM, HEX, ROX, CY5, and Quasar 705;

[0097] The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be invalid and must be repeated.

[0098] Positive judgment value

[0099] The Ct reference value of the target gene detected by this reagent was determined to be 38 through reference value research.

[0100] Interpretation of Test Results

[0101] 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.

[0102] Table 4. Interpretation of test results

[0103]

[0104] Example 3: Test results of the test samples of the composition of the present invention

[0105] The primers and probes shown in Example 1 were used to verify samples of Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, and Klebsiella oxytoca according to the method of Example 2. The results showed that the composition of the present invention can detect and distinguish any combination of the above-mentioned related pathogens. For example, the composition of the present invention detects:

[0106] Klebsiella aerogenes, Acinetobacter baumannii, Stenotrophomonas maltophilia, Klebsiella oxytoca ( Figure 1 );

[0107] Klebsiella aerogenes, Stenotrophomonas maltophilia, Klebsiella pneumoniae, Acinetobacter baumannii, Klebsiella oxytoca ( Figure 2 );

[0108] Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella oxytoca ( Figure 3 ).

[0109] These figures can prove that the present invention is a 6-plex PCR that can amplify the above-mentioned 6 targets at one time, and can be distinguished using a 6-channel PCR instrument. It also proves that any corresponding pair of combinations of the composition of the present invention can detect any corresponding combination of pathogens, and it can detect any combination of any corresponding 6 targets.

[0110] Example 4: Sensitivity of the composition of the present invention

[0111] Using the composition in Example 1 of the present invention, LOD (sensitivity) detection was performed on each target to simulate clinical samples and multiplex PCR detection was performed on a fluorescent quantitative PCR instrument. The results are as follows: Figure 4 As shown in Table 5, each channel can still accurately detect samples as low as 500 copies / mL, indicating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0112] Table 5

[0113]

[0114] Example 5. Specificity of the composition of the present invention

[0115] The composition of the present invention has no cross-reaction with common pathogens and other pathogens with similar infection symptoms (Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus, Enterococcus faecium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Streptococcus anginosus, Streptococcus mitis, Streptococcus intermedius). Figure 5 As shown, it is shown that the composition of the present invention has good specificity.

[0116] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0117] 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 ruled out 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. In order to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0118] Therefore, the inventors also designed some other primers and probes to form a different detection system 2 (the sequences are shown in Table 6, and the primer and probe sequences of Klebsiella pneumoniae in the replacement system) for the detection of the above pathogens. Figure 6 As shown in the figure, it can be seen that the detection performance of the six pathogen targets is reduced, the Ct value is delayed, and the fluorescence increment is reduced.

[0119] Table 6

[0120]

[0121]

Claims

1. A composition for detecting bloodstream infection-related pathogens, comprising upstream and downstream primers and probes for detecting at least 4 of the following related pathogens: Upstream and downstream primers and probes for detecting Klebsiella aerogenes as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Streptococcus anginosus as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 13 to 15; or The upstream and downstream primers and probes for detecting Klebsiella oxytoca are shown in SEQ ID NOs: 16-18.

2. The composition according to claim 1, characterized in that The composition comprises: Upstream and downstream primers and probes for detecting Klebsiella aerogenes as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Stenotrophomonas maltophilia as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Streptococcus anginosus as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Acinetobacter baumannii as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Klebsiella pneumoniae as shown in SEQ ID NOs: 13 to 16; and The upstream and downstream primers and probes for detecting Klebsiella oxytoca are shown in SEQ ID NOs: 16-18.

3. The composition according to claim 1, characterized in that The composition further comprises: Detect upstream and downstream primers and probes of internal standard.

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

5. The composition according to claim 4, characterized in that The fluorescent reporter group of the Klebsiella aerogenes probe is FAM; the fluorescent reporter group of the Stenotrophomonas maltophilia probe is HEX; the fluorescent reporter group of the Streptococcus anginosus probe is ROX; the fluorescent reporter group of the Acinetobacter baumannii probe is CY5; the fluorescent reporter group of the Klebsiella pneumoniae probe is Quasar 705; and the fluorescent reporter group of the Klebsiella oxytoca probe is ATTO425.

6. The composition according to any one of claims 1 to 5, characterized in that The components of the composition are present in admixture.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting pathogens associated with bloodstream infection, wherein: The type of the relevant pathogen is one or more of Klebsiella aerogenes, Stenotrophomonas maltophilia, Streptococcus anginosus, Acinetobacter baumannii, Klebsiella pneumoniae, or Klebsiella oxytoca.

8. A kit for detecting pathogens associated with bloodstream infection, the kit comprising the composition according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit also includes at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a nucleic acid amplifying reagent.

10. Use of a composition for preparing a reagent for detecting pathogens associated with bloodstream infection, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to claim 8 or 9; 3) Obtain and analyze the results.

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