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 staphylococcus intermedius, streptococcus angora, streptococcus intermedius, streptococcus constellati, streptococcus mitis and streptococcus oral cavity are detected and distinguished in a single-tube reaction system at the same time. Meanwhile, the detection sensitivity of the composition is higher and reaches 200 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 Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, and Streptococcus oralis. Background Art

[0002] Staphylococcus intermedius is a Gram-positive coccus belonging to the group of coagulase-positive Staphylococci (CoPS), originally isolated from the skin and respiratory tract of dogs and cats, and later also found in humans. The bacterium is spherical, usually arranged in pairs or short chains, and can form round, smooth, yellow colonies on regular culture media. It can ferment a variety of sugars, produce acid but no gas, and can produce coagulase. Staphylococcus intermedius is relatively common in the veterinary field and can cause skin, respiratory and urinary tract infections in animals. In humans, it is mainly associated with skin and soft tissue infections, wound infections, sepsis and endocarditis, etc. It can be transmitted by contact, especially close contact with pets, and may also be transmitted through medical equipment or hands.

[0003] Streptococcus anginosus is one of the normal flora colonized in the oral cavity, throat, digestive tract, etc., and is one of the Miller Streptococcus group. After scholars discovered Streptococcus anginosus in 1906, scholars have continuously reported the isolation of the bacteria 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 in vitro toxins, 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 Streptococcus anginosus bloodstream infection are that most patients have some underlying diseases, such as digestive system, cardiovascular system, respiratory system diseases and tumors. Many studies have shown that the characteristics of pharyngitis Streptococcus infection are not obvious and identification is difficult. In clinical work, if a related bacterial infection is suspected, specimens should be sent for testing in a timely manner, antibiotics should be used reasonably based on drug sensitivity results, and drug resistance monitoring should be performed to improve prognosis.

[0004] Streptococcus intermedius is a Gram-positive bacterium in the genus Streptococcus and belongs to the oral streptococcus group. The bacterium is a facultative anaerobe that can grow under aerobic and anaerobic conditions. It is usually spherical or ellipsoidal in shape and arranged in pairs or short chains. The bacterium can produce a small amount of α-hemolysis or no hemolysis, which helps to distinguish it from other streptococci. Streptococcus intermedius is part of the normal human flora and is mainly found in the oral cavity, digestive tract and reproductive tract. However, when the body's immunity is reduced or the local defense mechanism is impaired, it can become an opportunistic pathogen and cause a variety of infections, including brain abscess, liver abscess, abdominal infection, osteomyelitis and soft tissue infection. Laboratory diagnosis is usually based on microbial culture and biochemical identification, and molecular biological methods such as PCR can also be used for rapid detection. In short, although Streptococcus intermedius is a normal commensal bacterium in healthy humans, it can cause serious infections under certain conditions, so it is very important to recognize and treat it in clinical practice.

[0005] Streptococcus constellatus is a Gram-positive coccus belonging to group D of the genus Streptococcus. Together with Streptococcus intermedius and Streptococcus anginosus, it forms the Anginosus group. This bacterium is spherical or oval in shape, usually arranged in pairs or short chains. It can grow on blood-containing culture media to form small, transparent colonies, and α-hemolysis or γ-hemolysis can sometimes be seen. Streptococcus constellatus is part of the normal human flora and is mainly found in the oral cavity, digestive tract, and reproductive tract. However, when the body's immunity is reduced or local defense mechanisms are impaired, it can become an opportunistic pathogen and cause a variety of infections, including brain abscesses, liver abscesses, abdominal infections, osteomyelitis, and soft tissue infections. Infections with Streptococcus constellatus usually manifest as acute or chronic inflammation, with diverse clinical symptoms and varying degrees of severity.

[0006] 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.

[0007] Streptococcus oralis is a gram-positive coccus belonging to the Anginosus group of the Streptococcus genus and is part of the normal oral flora of the human body. This bacterium is commonly found in the oral mucosa, tooth surfaces and saliva and is associated with a variety of oral health conditions. Oral Streptococcus is generally considered a conditional pathogen and is usually harmless in healthy individuals, but in certain circumstances, such as when the immune system is impaired or other infections are present, it can cause local or systemic infections. These infections may include periodontal disease, apical periodontitis, and more serious diseases such as endocarditis and sepsis. The bacterium is able to produce a variety of enzymes and metabolites that aid its colonization and survival in the oral cavity. Laboratory diagnosis is usually performed by bacterial culture and biochemical identification, and molecular biological methods such as PCR can also be used for rapid detection. Treatment is usually antibiotic therapy, and commonly used antibiotics include penicillin, vancomycin, and clindamycin, etc. The specific choice needs to be determined based on the results of drug sensitivity tests. Understanding its biological characteristics and drug resistance is essential for the effective prevention and treatment of infections caused by oral Streptococcus.

[0008] The above-mentioned Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, and Streptococcus oralis 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 Staphylococcus intermedius as shown in SEQ ID NOs: 1 to 3;

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

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

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

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

[0016] The upstream and downstream primers and probes for detecting oral streptococci 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 four pathogens among Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, and Streptococcus oralis 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 200 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 Staphylococcus intermedius as shown in SEQ ID NOs: 1 to 3;

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

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

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

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

[0025] The upstream and downstream primers and probes for detecting oral streptococci 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 Staphylococcus intermedius probe is FAM; the fluorescent reporter group of the Streptococcus anginosus probe is HEX; the fluorescent reporter group of the Streptococcus intermedius probe is ROX; the fluorescent reporter group of the Streptococcus constellatus probe is CY5; the fluorescent reporter group of the Streptococcus mitis probe is Quasar 705; and the fluorescent reporter group of the Streptococcus oralis 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 Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, and Streptococcus oralis.

[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 DEPC H 2 O, physiological saline, and at least one of the internal standard genes. The positive quality control product is at least one of the plasmid or fragment genes of Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, and Streptococcus oralis.

[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 consists of Tris-HCl, MgCl 2 , KCl, Triton X-100, etc. The total volume of a single PCR reaction tube is generally 20μl to 200μl.

[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-mentioned pathogens and suffers from the infection. For example, the method can detect whether the environment and culture have the above-mentioned 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 Staphylococcus intermedius probe is FAM; the fluorescent reporter group of the Streptococcus anginosus probe is HEX; the fluorescent reporter group of the Streptococcus intermedius probe is ROX; the fluorescent reporter group of the Streptococcus constellatus probe is CY5; the fluorescent reporter group of the Streptococcus mitis probe is Quasar 705; and the fluorescent reporter group of the Streptococcus oralis 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[ddH 2 The]]> 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]

[0105] Example 3: Test results of the test samples of the composition of the present invention

[0106] The primers and probes shown in Example 1 were verified according to the method of Example 2 on samples of Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, and Streptococcus oralis. 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:

[0107] Streptococcus anginosus, Streptococcus constellatus, Streptococcus intermedius, Streptococcus mitis ( Figure 1 );

[0108] Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis ( Figure 2 );

[0109] Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, Streptococcus oralis ( Figure 3 ).

[0110] 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.

[0111] Example 4: Sensitivity of the composition of the present invention

[0112] 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 200 copies / mL, indicating that the sensitivity of the composition of the present invention is 200 copies / mL.

[0113] Table 5

[0114]

[0115] Example 5. Specificity of the composition of the present invention

[0116] The composition of the present invention has no cross reaction with common pathogens and other pathogens with similar infection symptoms (Enterococcus faecalis, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Yersinia intestinalis, Shigella, Campylobacter, Escherichia coli O157, Clostridium difficile, Clostridium perfringens, Helicobacter pylori, enterovirus 71, astrovirus, norovirus, rotavirus, enteroadenovirus). The results are as follows Figure 5 As shown, it is shown that the composition of the present invention has good specificity.

[0117] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0118] 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.

[0119] 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 oral streptococci in the system are replaced), which is also used to detect 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.

[0120] Table 6

[0121]

[0122]

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 Staphylococcus intermedius as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Streptococcus anginosus as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Streptococcus intermedius as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Streptococcus constellatus as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Streptococcus mitis as shown in SEQ ID NOs: 13 to 15; or The upstream and downstream primers and probes for detecting oral streptococci 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 Staphylococcus intermedius as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Streptococcus anginosus as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Streptococcus intermedius as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Streptococcus constellatus as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Streptococcus mitis as shown in SEQ ID NOs: 13 to 16; and The upstream and downstream primers and probes for detecting oral streptococci 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 Staphylococcus intermedius probe is FAM; the fluorescent reporter group of the Streptococcus anginosus probe is HEX; the fluorescent reporter group of the Streptococcus intermedius probe is ROX; the fluorescent reporter group of the Streptococcus constellatus probe is CY5; the fluorescent reporter group of the Streptococcus mitis probe is Quasar 705; and the fluorescent reporter group of the Streptococcus oralis 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 Staphylococcus intermedius, Streptococcus anginosus, Streptococcus intermedius, Streptococcus constellatus, Streptococcus mitis, or Streptococcus oralis.

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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