A combination for simultaneously detecting five pathogens of bloodstream infection

Abstract

The present invention belongs to the technical field of in vitro nucleic acid detection in molecular biology, and specifically, relates to a composition for simultaneously detecting 5 pathogens of bloodstream infection; more specifically, it relates to a composition, a kit and its use for detecting and distinguishing Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus. The joint detection composition provided by the present invention uses a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby realizing rapid detection of target genes of at least 3 pathogens in a single-tube reaction system, and having a higher detection sensitivity of 200 copies / mL. The present invention can accurately detect pathogens, and can also analyze the mixed infection of each pathogen, so as to achieve the effect of precise medication, reduce the use of antibiotics, and save costs. Provide clinicians with a more sufficient basis for rapid diagnosis and accelerate the implementation of treatment measures for patients.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro nucleic acid detection in molecular biology, and specifically, to a composition, a kit and uses thereof for simultaneously detecting multiple pathogens of bloodstream infection; more specifically, to a composition, a kit and uses thereof for simultaneously detecting Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus infected with bloodstream. Background Art

[0002] Staphylococcus lugdunensis ( Staphylococcus lugdunensis ) is a Gram-positive, spherical bacterium belonging to the genus Staphylococcus that is commonly found in the normal flora of the skin, nasal cavity, and upper respiratory tract of healthy people. Although it is usually harmless, as an opportunistic pathogen, it may cause serious infections in patients with compromised immune function or a history of surgery. The bacterium can cause a variety of infections, the most common of which is endocarditis, especially in patients with prosthetic heart valves or other cardiac devices. It may also cause skin and soft tissue infections, arthritis, osteomyelitis, and urinary tract infections. Staphylococcus lugdunensis is beta-hemolytic or alpha-hemolytic on culture media, and its coagulase test is usually negative, but other biochemical characteristics are similar to common staphylococci. Although it may be resistant to some antibiotics (such as methicillin), many strains are sensitive to drugs such as vancomycin and rifampicin. Treatment usually relies on antibiotics, and the treatment plan is adjusted according to antibiotic sensitivity testing. Due to its high pathogenicity in serious infections such as endocarditis and its resistance to certain antibiotics, special attention should be paid to the detection and treatment of this bacterium in clinical practice.

[0003] Staphylococcus intermedius ( Staphylococcus intermedius ) is a Gram-positive coccus belonging to the group of coagulase-positive Staphylococci (CoPS), which was originally isolated from the skin and respiratory tract of dogs and cats and was later found in humans. This 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 system 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 through contact, especially close contact with pets, and may also be transmitted through medical equipment or hands. Laboratory diagnosis is usually based on bacterial culture and biochemical identification, such as coagulase test and catalase test, and molecular biological methods such as PCR can also be used for rapid detection. Antibiotic therapy is usually used for treatment. Commonly used antibiotics include vancomycin, daptomycin and linezolid. The specific choice should be determined based on the results of drug sensitivity tests. Understanding its biological characteristics and drug resistance is essential for effective diagnosis and treatment.

[0004] Streptococcus agalactiae ( Streptococcus agalactiae, GBS (GBS for short) is a Gram-positive coccus that belongs to Group B in serology, so it is also called Group B Streptococcus. It is the leading pathogen of neonatal infection, causing a high infection rate and mortality rate in neonates. Streptococcus agalactiae is an important cause of postpartum sepsis in pregnant women and neonatal meningitis. GBS normally resides in women's vagina and human intestines. 30% of healthy people's feces and 25% of healthy people's urogenital system carry this bacteria. The vaginal carriage rate of non-pregnant women is 10.56%. When the human immune function is reduced, it will provide opportunities for infection with Streptococcus agalactiae. During pregnancy, GBS can cause chorioamnionitis in parturients, leading to miscarriage, premature rupture of membranes and intrauterine infection. During the delivery process, newborns pass through the mother's GBS-contaminated birth canal, which can lead to pneumonia, meningitis, sepsis, etc. after birth.

[0005] Listeria monocytogenes ( Listeria monocytogenes ) is a Gram-positive, non-spore-forming, highly motile bacterium belonging to the genus Listeria. It is a pathogen that is widely present in the environment and can grow at refrigerated temperatures, making it an important pathogen in food safety. Listeria monocytogenes can be transmitted through food, especially raw food, undercooked meat, cheese, etc. It can enter the human body through the digestive tract and spread to different organs through the blood or lymph, causing systemic infection. The main pathogenic mechanism of this bacterium is that it can escape the host's immune surveillance by invading host cells and growing and multiplying inside the cells. Infection usually manifests as listeriosis, with the main symptoms including fever, muscle pain, gastrointestinal discomfort, etc. Severe patients may develop meningitis and sepsis, which is particularly harmful to pregnant women, newborns, the elderly and immunosuppressed patients. Infection in pregnant women may cause miscarriage, premature birth or neonatal infection. Listeria monocytogenes has a strong tolerance to conventional food processing methods and can grow in low-temperature, low-oxygen environments, so special attention needs to be paid to control in the food industry. Antibiotic treatment is usually effective, but due to its drug resistance problem, early diagnosis and timely treatment are essential.

[0006] Streptococcus constellatus Streptococcus constellatus ) is a Gram-positive coccus belonging to group D of the genus Streptococcus. Together with Streptococcus intermedius and Streptococcus anginosus, it constitutes the group Streptococcus anginosus ( Anginosus group). This bacterium is spherical or oval, usually arranged in pairs or short chains, and can grow on blood-containing culture media to form small, transparent colonies, and α-hemolysis or γ-hemolysis can sometimes be seen. Streptococcus australis 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 damaged, it can become an opportunistic pathogen, causing a variety of infections, including brain abscesses, liver abscesses, abdominal infections, osteomyelitis, and soft tissue infections. Infections with Streptococcus australis usually manifest as acute or chronic inflammation, with diverse clinical symptoms and varying degrees of severity. Laboratory diagnosis mainly relies on bacterial culture and biochemical identification, such as coagulase test and catalase test. Molecular biological methods such as PCR can also be used for rapid detection.

[0007] The above-mentioned Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes, and Streptococcus constellatus 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.

[0008] Therefore, there is an urgent need in this field for a simple, rapid and objective method for detecting the above pathogens, so as to identify the pathogens, diagnose them early and reasonably guide antibiotic treatment. Summary of the invention

[0009] In view of the above-mentioned shortcomings, in the first aspect, the present invention provides a composition for simultaneously detecting multiple pathogens of bloodstream infection, wherein the composition comprises at least three groups of primers and probes for detecting the following pathogens:

[0010] Upstream primers, downstream primers and probes for detecting Staphylococcus lugdunensis as shown in SEQ ID NOs: 1 to 3;

[0011] Upstream primers, downstream primers and probes for detecting Staphylococcus intermedius as shown in SEQ ID NOs: 4 to 6;

[0012] Upstream primers, downstream primers and probes for detecting Streptococcus agalactiae as shown in SEQ ID NOs: 7 to 9;

[0013] Upstream primers, downstream primers and probes for detecting Listeria monocytogenes as shown in SEQ ID NOs: 10 to 12;

[0014] The upstream primers, downstream primers and probe for detecting Streptococcus constellatus are shown in SEQ ID NOs: 13-15.

[0015] The composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to upstream primers, downstream primers and probes that match each other for detecting a target.

[0016] The composition of the present invention can be combined into any combination of detection corresponding to 5 pathogen 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.

[0017] For example, it may include any 4 groups of primers and probes for detecting the above 5 groups of pathogens; it may include any 3 groups of primers and probes for detecting the above 5 groups of pathogens; it may include any 2 groups of primers and probes for detecting the above 5 groups of pathogens; it may include any 1 group of primers and probes for detecting the above 5 groups of pathogens.

[0018] The above-mentioned joint inspection composition provided by the present invention uses 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 3 pathogens among Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus in a single-tube reaction system. The composition of the present invention has a higher detection sensitivity of 200 copies / mL. The present invention can accurately detect pathogens and can also analyze the mixed infection of each pathogen, thereby achieving the effect of precise medication, reducing the use of antibiotics and saving costs. It provides clinicians with a more sufficient basis for rapid diagnosis and accelerates the implementation of treatment measures for patients.

[0019] Furthermore, in the composition, the fluorescent reporter groups of the probes are different from each other and do not interfere with each other.

[0020] Furthermore, in the present invention, "different from each other and not interfering with each other" means that the fluorescent reporter groups used by each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO 425, ATTO 700, Quasar 705, FAM, VIC, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0021] In a specific embodiment, the fluorescent reporter group of the probe for detecting Staphylococcus lugdunensis as shown in SEQ ID NO: 3 is FAM; the fluorescent reporter group of the probe for detecting Staphylococcus intermedius as shown in SEQ ID NO: 6 is HEX; the fluorescent reporter group of the probe for detecting Streptococcus agalactiae as shown in SEQ ID NO: 9 is ROX; the fluorescent reporter group of the probe for detecting Listeria monocytogenes as shown in SEQ ID NO: 12 is CY5; the fluorescent reporter group of the probe for detecting Streptococcus constellatus as shown in SEQ ID NO: 15 is Quasar 705.

[0022] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1, BHQ2, or MGB.

[0023] In a specific embodiment, the 3' end of the probe is BHQ1.

[0024] In a specific embodiment, the 3' end of the probe is BHQ2.

[0025] Furthermore, the composition exists in a mixed form.

[0026] Furthermore, the dosage of a single primer in the composition is 0.01µM~0.5µM; the dosage of a single probe in the composition is 0.005µM~0.2µM.

[0027] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0028] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0029] In a second aspect, the present invention also provides the use of the above-mentioned composition in the preparation of a kit for simultaneously detecting multiple pathogens of bloodstream infection, wherein the multiple pathogens include at least three of Staphylococcus agalactiae, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes, and Streptococcus constellatus.

[0030] Furthermore, the kit is a fluorescent PCR detection kit.

[0031] A third aspect: The present invention also provides a kit for simultaneously detecting multiple pathogens of bloodstream infection, the kit comprising the above-mentioned composition.

[0032] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, UNG enzyme, PCR buffer, Taq enzyme, Mg 2+ , dNTP.

[0033] Furthermore, the PCR buffer is composed of Tris-HCl, MgCl 2 , KCl, Triton X-100 and other buffer systems.

[0034] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0035] In a specific embodiment, the negative quality control is at least one of DEPC water and sterile saline. The positive quality control is at least one of a fragment plasmid or a fragment DNA or a fragment RNA of a specific gene target sequence of Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus.

[0036] In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0037] In a fourth aspect, the present invention also provides a method for simultaneously detecting multiple pathogens of bloodstream infection for non-diagnostic purposes, the method comprising the following steps:

[0038] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0039] 2) using any of the above compositions or any of the above kits to perform fluorescent quantitative PCR analysis on the nucleic acid obtained in step 1);

[0040] 3) Obtain and analyze the results.

[0041] 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 bloodstream infection, etc. For example, the presence of the above pathogens can be detected in a test culture (such as blood, etc.).

[0042] Furthermore, the reaction conditions of the fluorescent quantitative PCR are: UNG enzyme reaction, 50°C, 5 minutes, 1 cycle; Taq enzyme activation, 95°C, 1 minute, 1 cycle; denaturation, 95°C, 10 seconds, annealing, extension and fluorescence collection, 60°C, 20 seconds, 41 cycles.

[0043] Detection principle of this application: The most common method for pathogen detection is fluorescent quantitative polymerase chain reaction (PCR for short). In the PCR reaction system, a pair of specific primers and a hydrolysis probe (Taqman probe) are included. The probe is a specific oligonucleotide sequence, and the two ends are labeled with a fluorescent reporter group (5' end) and a fluorescent quencher group (3' end). When the probe is complete, the fluorescent signal emitted by the fluorescent reporter group is absorbed by the fluorescent 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 fluorescent reporter group is separated from the fluorescent quencher group to emit fluorescence. For each amplification of a DNA chain, a fluorescent molecule will be 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 nucleic acid concentration of the pathogen. The higher the concentration of the pathogen nucleic acid, the smaller the Ct value. Finally, the infection can be judged based on the Ct value. At the same time, the UNG enzyme + dUTP anti-pollution system used in this reagent ensures the accuracy of the detection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a graph showing the test results of the five-combination test of the composition of the present invention;

[0045] Figure 2 This is a diagram showing the test results of the quadruple test of the composition of the present invention;

[0046] Figure 3 This is a diagram of the test results of the triple test of the composition of the present invention;

[0047] Figure 4 The figure is a test result diagram of the sensitivity of the composition of the present invention;

[0048] Figure 5 This is a graph showing the specificity of the composition of the present invention;

[0049] Figure 6 This is a graph showing the results of a joint test of the composition of Comparative Example 1 of the present invention on Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus. DETAILED DESCRIPTION

[0050] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0051] Example 1: Primers and probes used in the present invention

[0052] The primers and probes used in the present invention are shown in Table 1 below:

[0053] Table 1:

[0054]

[0055] Among them, the fluorescent reporter group of the probe for detecting Staphylococcus lugdunensis is FAM; the fluorescent reporter group of the probe for detecting Staphylococcus intermedius is HEX; the fluorescent reporter group of the probe for detecting Streptococcus agalactiae is ROX; the fluorescent reporter group of the probe for detecting Listeria monocytogenes is CY5; and the fluorescent reporter group of the probe for detecting Streptococcus constellatus is Quasar 705.

[0056] Example 2: Method for Simultaneous Detection of Multiple Pathogens of Bloodstream Infection

[0057] (1) Fluorescence PCR amplification reaction solution: contains PCR buffer, Taq enzyme, UNG enzyme, Mg 2+ , dNTP, primers, probes, etc. The specific reaction system is shown in Table 2:

[0058] Table 2:

[0059]

[0060] The enzyme mix is ​​a mixture of UNG enzyme (2U / µL) and H-Taq enzyme (DNA polymerase) (5U / µL) at a volume ratio of 2:8. The above PCR buffer is composed of Tris-HCl, MgCl 2, KCl, Triton X-100 and other buffer systems; the sample DNA in this embodiment is the sample to be tested obtained by extracting nucleic acid from the sample to be tested using a commercially available nucleic acid extraction or purification reagent according to its instructions; the sample to be tested in this embodiment is blood. When doing a negative control experiment, the sample to be tested is replaced with sterile physiological saline; when doing a positive control, the sample to be tested is replaced with a mixed plasmid containing the corresponding specific gene target sequence.

[0061] (2) The amplification reaction procedure is shown in Table 3:

[0062] Table 3:

[0063]

[0064] (3) Result analysis and judgment:

[0065] 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.

[0066] Quality Control:

[0067] Negative control: no Ct value is displayed in the curves of the five channels of FAM, HEX, ROX, CY5, and Quasar 705;

[0068] Positive control: Ct≤36 for the five channels of FAM, HEX, ROX, CY5, and Quasar 705;

[0069] The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be invalid and must be repeated.

[0070] Positive judgment value:

[0071] The Ct reference value of the target gene detected by this reagent was determined to be 38 through reference value research.

[0072] Explanation of test results:

[0073] If the FAM, HEX, ROX, CY5 and Quasar 705 channels have obvious S-shaped amplification curves and the Ct value is ≤38, it is judged as positive; if the FAM, HEX, ROX, CY5 and Quasar 705 channels have no amplification curve (No Ct) or the Ct value is greater than 38, it is judged as negative. The details are shown in Table 4:

[0074] Table 4:

[0075]

[0076] Example 3: Test results of the test samples of the composition of the present invention

[0077] The primers and probes shown in Example 1 were used to perform joint testing and differentiation on pathogen-positive mixed samples of Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus according to the method of Example 2. The test results are as follows: Figure 1 As shown. Figure 1 It can be seen that the composition of the present application can simultaneously detect and differentiate Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus.

[0078] Example 4, quadruple test results of the composition of the present invention

[0079] The primers and probes shown in Example 1 were used to perform joint testing and differentiation on pathogen-positive mixed samples of Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae and Listeria monocytogenes according to the method of Example 2. The test results are as follows: Figure 2 As shown. Figure 2 It can be seen that the composition of the present application can also perform quadruple detection and differentiate Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae and Listeria monocytogenes.

[0080] Example 5. Triple test results of the composition of the present invention

[0081] The primers and probes shown in Example 1 were used to perform joint testing and differentiation on pathogen-positive mixed samples of Staphylococcus lugdunensis, Staphylococcus intermedius and Streptococcus agalactiae according to the method of Example 2. The test results are as follows: Figure 3 As shown. Figure 3 It can be seen that the composition of the present application can also perform triple detection and differentiation on Staphylococcus lugdunensis, Staphylococcus intermedius and Streptococcus agalactiae.

[0082] Example 6: Sensitivity of the composition of the present invention

[0083] The positive samples were diluted with negative samples (sterile saline) to a concentration of 200 copies / mL, and the test was repeated 20 times to verify the sensitivity of the composition and kit of the present invention. Figure 4As shown in Table 5 below, the test results show that the composition can still accurately detect samples below 200 copies / mL in each channel. The results show that the composition of the present invention has high sensitivity and the detection concentration can reach 200 copies / mL.

[0084] Table 5:

[0085]

[0086] Example 7. Specificity of the composition of the present invention

[0087] The primers and probes shown in Example 1 were used in accordance with the method described in Example 2 to perform multiplex PCR detection on a fluorescent quantitative PCR instrument for other bloodstream infection pathogens (Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus, Enterococcus faecium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Streptococcus anginosus, Streptococcus mitis, Streptococcus intermedius, Norovirus, rotavirus, and enteric adenovirus) that have homology to nucleic acid sequences and are likely to cause the same or similar clinical symptoms. The results are shown in the following table. Figure 5 As shown, from Figure 5 It can be seen that the detection of each target point is negative, which proves that the composition of the present invention has good specificity.

[0088] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0089] 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. To ensure the conservatism of the design (conservatism is crucial to the accuracy of the test), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0090] Therefore, the inventors also designed some other primers and probes, such as another pair of upstream primers, downstream primers and probes for Streptococcus constellatus (see Table 6 and SEQ ID NOs: 16-18 in the sequence table), which were replaced with the upstream primers, downstream primers and probes for Streptococcus constellatus in the kit of the present invention, and the replaced detection system was used to perform a joint test on pathogen-positive mixed samples of Staphylococcus lugdunensis, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes and Streptococcus constellatus. The results are as follows: Figure 6 As shown. Figure 6 and Figure 1 From the comparison of the amplification curves, it can be seen that when the comparative example composition is used for joint detection, the performance of Streptococcus constellatus is reduced, the Ct value is delayed, and the fluorescence increment decreases. This shows that the combination of primers and probes in the present invention is irreplaceable.

[0091] Table 6:

[0092]

[0093] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A composition for simultaneously detecting multiple pathogens of bloodstream infection, characterized in that: The composition includes at least 3 sets of primers and probes for detecting the following pathogens: Upstream primers, downstream primers and probes for detecting Staphylococcus lugdunensis as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Staphylococcus intermedius as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting Streptococcus agalactiae as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting Listeria monocytogenes as shown in SEQ ID NOs: 10 to 12; The upstream primers, downstream primers and probe for detecting Streptococcus constellatus are shown in SEQ ID NOs: 13-15.

2. The composition for simultaneously detecting multiple pathogens of bloodstream infection according to claim 1, characterized in that: In the composition, the fluorescent reporter groups of the probes are different from each other and do not interfere with each other.

3. The composition according to claim 2, characterized in that The fluorescent reporter group of the probe for detecting Staphylococcus lugdunensis as shown in SEQ ID NO: 3 is FAM; The fluorescent reporter group of the probe for detecting Staphylococcus intermedius as shown in SEQ ID NO: 6 is HEX; The fluorescent reporter group of the probe for detecting Streptococcus agalactiae as shown in SEQ ID NO: 9 is ROX; The fluorescent reporter group of the probe for detecting Listeria monocytogenes as shown in SEQ ID NO: 12 is CY5; The fluorescent reporter group of the probe for detecting Streptococcus constellatus as shown in SEQ ID NO: 15 is Quasar 705.

4. The composition according to claim 1, characterized in that The composition is present in a mixed form.

5. Use of the composition according to any one of claims 1 to 4 in preparing a kit for simultaneously detecting multiple pathogens of bloodstream infection, characterized in that: The plurality of pathogens include at least three of Staphylococcus agalactiae, Staphylococcus intermedius, Streptococcus agalactiae, Listeria monocytogenes, and Streptococcus constellatus.

6. The use according to claim 5, characterized in that The kit is a fluorescent PCR detection kit.

7. A kit for simultaneously detecting multiple pathogens of bloodstream infection, characterized in that: The kit comprises the composition according to any one of claims 1 to 4.

8. The kit according to claim 7, characterized in that The kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a Taq enzyme, a Mg 2+ , UNG enzyme, and at least one of dNTP.

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

10. A method for simultaneously detecting multiple pathogens of 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 analysis 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 7 to 9; 3) Obtain and analyze the results.

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