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 aureus, staphylococcus capitis, bacteroides fragilis, streptococcus agalactiae, streptococcus salivarius and staphylococcus hominis 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 in particular, relates to a composition for detecting and distinguishing pathogens related to bloodstream infection, including Staphylococcus aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, and Staphylococcus hominis. Background Art

[0002] Staphylococcus aureus is a Gram-positive aerobic bacterium. It is the most pathogenic among the genus Staphylococcus. It mainly causes local suppurative infections, and can also cause pneumonia, infective endocarditis and osteomyelitis, and even sepsis and septicemia. Staphylococcus aureus can produce coagulase to coagulate blood. It has strong resistance to antibiotics, low sensitivity to sulfonamide drugs, but high sensitivity to penicillin and erythromycin. Staphylococcus aureus is widely present in nature and can be detected in air, water, dust, and human and animal excrement. It is a conditional pathogen and one of the common pathogens of community and hospital infections. According to the US Centers for Disease Control and Prevention, the infection caused by Staphylococcus aureus ranks second, second only to Escherichia coli. As one of the most threatening bacteria to human and animal health, Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus (MRSA), carries a large number of virulence factors, can cause widespread infection, and is one of the key pathogens causing public health safety issues.

[0003] Staphylococcus capitis is a common coagulase-negative staphylococcus, named because it is often isolated from the head, face, and neck of humans. Staphylococcus capitis is a Gram-positive bacterium with a cocci-shaped microscopic shape and arranged in a V or W shape. Staphylococcus capitis is detected more frequently in sputum and secretions, and the disease types are mainly lung infection and wound infection. As a conditional pathogen, this bacterium can cause various serious infections, such as mastitis, complicated skin and soft tissue infection, artificial joint infection, endocarditis, pacemaker-related infection, neonatal hospital-acquired delayed sepsis, etc. Staphylococcus capitis has more than 3 types of drug resistance, which is a multidrug-resistant bacterium and should be taken seriously in clinical practice. Staphylococcus capitis is mostly a patient with low immunity in patients with bloodstream infection. The genus, which is a conditional pathogen, may become a pathogen in this population, so it cannot be ignored in clinical treatment.

[0004] Bacteroides fragilis is a commensal bacterium that colonizes the intestines of mammals and is also a common conditional pathogen in clinical infection cases. It can decompose sugar and tolerate bile, and is a representative strain of the genus Bacteroides. Bacteroides fragilis is a Gram-negative bacillus that can maintain the balance of the digestive system and the body's resistance under normal circumstances. Bacteroides fragilis often parasitizes in the human oral cavity, intestines and female reproductive tract. It is a conditional pathogen that is mainly endogenous infection and can cause female reproductive system, chest and intracranial infections.

[0005] Streptococcus agalactiae (GBS) 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.

[0006] Streptococcus salivarius is a Gram-positive spherical bacterium that mainly colonizes the human oral cavity and intestines. In the digestive tract and oral ecosystem, the bacterium plays an important role in establishing immune homeostasis. It is not the bacterium that causes most tooth decay, but the bacterium can cause sepsis in patients with neutropenia. Certain procedures that may cause trauma, such as dental work or improper brushing, may cause microorganisms to enter the patient's blood. Streptococcus salivarius does not generally have the potential to be a highly toxic organism. However, in immunosuppressed patients, its role as a pathogenic microorganism has been established, especially in cancer patients and patients with cirrhosis. In patients with cirrhosis, Streptococcus salivarius has produced infections associated with invasive surgical procedures, such as endoscopic ligation of esophageal varices. Therefore, it has been found that the bacteria can cause bacteremia and cellulitis in patients with cirrhosis.

[0007] Staphylococcus hominis belongs to the third type of coagulase-negative staphylococci (CoNS). It is considered a potential pathogen due to its ability to form biofilms on smooth surfaces of human body devices. It can cause different infections such as infective endocarditis and meningitis in immunocompromised patients. It is usually isolated from specimens of patients with hospital-acquired infections. The formation of stable biofilms on medical devices or host tissues is considered to be its main pathogenic factor. However, Staphylococcus hominis is a rare conditional pathogen that can cause opportunistic infections in various parts of the body only when the body's immunity is low. Therefore, it is one of the three most common isolates in the blood of newborns and immunosuppressed patients, and is considered to be the cause of bacteremia, sepsis and endocarditis.

[0008] The above-mentioned Staphylococcus aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, and Staphylococcus hominis 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] At present, the commonly used single-tube multiplex detection system mostly adopts the melting curve method. By selecting different fluorescent signals, it can realize the detection of dozens of pathogens in a single tube, but the sensitivity of this technology is low, because the melting curve method relies on the large accumulation of double-stranded DNA and the analysis of nucleic acid by detecting its melting behavior. If the initial template amount is small, the signal change is not obvious in the early accumulation of double-stranded DNA. Only when the template amount reaches a certain level can the obvious signal change be detected in the melting process, and it relies on non-specific fluorescent dyes to detect the total amount of all double-stranded DNA, including non-specific products. Therefore, relatively speaking, its detection sensitivity for low-concentration nucleic acid samples is not as good as that of the probe method, and the sensitivity of the melting curve method is difficult to reach within 3000 copies / mL. The pathogen load contained in the blood of patients with bloodstream infection is very low, generally not more than 500 copies / mL, and direct extraction of pathogens from the blood for detection requires very high sensitivity. Therefore, in theory, it is impossible to detect from the blood using the melting curve, and the fluorescent PCR method uses specific probes to only mark and detect the target sequence, so it is more sensitive. The advantages of fluorescent PCR are high specificity and sensitivity. Therefore, the fluorescent PCR method can be used to directly detect pathogens in the blood.

[0010] 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

[0011] 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 related pathogens:

[0012] Upstream and downstream primers and probes for detecting Staphylococcus aureus as shown in SEQ ID NOs: 1 to 3;

[0013] Upstream and downstream primers and probes for detecting Staphylococcus capitis as shown in SEQ ID NOs: 4 to 6;

[0014] Upstream and downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NOs: 7 to 9;

[0015] Upstream and downstream primers and probes for detecting Streptococcus agalactiae as shown in SEQ ID NOs: 10 to 12;

[0016] Upstream and downstream primers and probes for detecting Streptococcus salivarius as shown in SEQ ID NOs: 13 to 15; or

[0017] The upstream and downstream primers and probes for detecting Staphylococcus aureus are shown in SEQ ID NOs: 16-18.

[0018] 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 aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, and Staphylococcus hominis 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 preventive policies in a timely manner.

[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 aureus as shown in SEQ ID NOs: 1 to 3;

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

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

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

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

[0025] The upstream and downstream primers and probes for detecting Staphylococcus aureus 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.

[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 aureus probe is FAM; the fluorescent reporter group of the Staphylococcus capitis probe is HEX; the fluorescent reporter group of the Bacteroides fragilis probe is ROX; the fluorescent reporter group of the Streptococcus agalactiae probe is CY5; the fluorescent reporter group of the Streptococcus salivarius probe is Quasar 705; and the fluorescent reporter group of the Staphylococcus hominis 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 a 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 aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, and Staphylococcus hominis.

[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 aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, and Staphylococcus hominis.

[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 6It 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 aureus probe is FAM; the fluorescent reporter group of the Staphylococcus capitis probe is HEX; the fluorescent reporter group of the Bacteroides fragilis probe is ROX; the fluorescent reporter group of the Streptococcus agalactiae probe is CY5; the fluorescent reporter group of the Streptococcus salivarius probe is Quasar 705; and the fluorescent reporter group of the Staphylococcus hominis 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]

[0094] 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.

[0095] Quality Control

[0096] Negative control: no Ct value is displayed in the curves of the three channels of ATTO425, FAM, HEX, ROX, CY5, and Quasar 705;

[0097] Positive control: Ct≤36 for the three channels of ATTO425, FAM, HEX, ROX, CY5, and Quasar 705;

[0098] The above requirements must be met simultaneously in the same experiment, otherwise, the experiment will be invalid and must be repeated.

[0099] Positive judgment value

[0100] The Ct reference value of the target gene detected by this reagent was determined to be 38 through reference value research.

[0101] Interpretation of Test Results

[0102] 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.

[0103] Table 4. Interpretation of test results

[0104]

[0105]

[0106] Example 3: Test results of the test samples of the composition of the present invention

[0107] The primers and probes shown in Example 1 were verified according to the method of Example 2 on samples of Staphylococcus aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, and Staphylococcus hominis. 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:

[0108] Staphylococcus aureus, Streptococcus agalactiae, Bacteroides fragilis, Streptococcus capitis ( Figure 1 );

[0109] Staphylococcus aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Staphylococcus hominis ( Figure 2 );

[0110] Staphylococcus aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, Staphylococcus hominis ( Figure 3 ).

[0111] These figures prove that the present invention is a 6-plex PCR that can amplify the above 6 targets at one time and can be distinguished using a 6-channel PCR instrument. It also proves that any corresponding pair combination of the composition of the present invention can detect any corresponding combination of pathogens.

[0112] Example 4: Sensitivity of the composition of the present invention

[0113] 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.

[0114] Table 5

[0115]

[0116]

[0117] Example 5. Specificity of the composition of the present invention

[0118] The composition of the present invention has no cross reaction with common pathogens and other pathogens with similar infection symptoms (Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter aerogenes, Serratia marcescens, Stenotrophomonas maltophilia, Candida albicans, Klebsiella oxytoca, Streptococcus anginosus, Streptococcus mitis, Burkholderia cepacia, Candida glabrata, Candida parapsilosis, Candida tropicalis, Aspergillus, Mucor, Cryptococcus neoformans, Pneumocystis jiroveci). The results are as follows Figure 5 As shown, it is shown that the composition of the present invention has good specificity.

[0119] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0120] 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.

[0121] 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 Streptococcus salivarius in the system are replaced), which are 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.

[0122] Table 6

[0123]

[0124]

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 aureus as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Staphylococcus capitis as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Streptococcus agalactiae as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Streptococcus salivarius as shown in SEQ ID NOs: 13 to 15; or The upstream and downstream primers and probes for detecting Staphylococcus aureus 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 aureus as shown in SEQ ID NOs: 1 to 3; Upstream and downstream primers and probes for detecting Staphylococcus capitis as shown in SEQ ID NOs: 4 to 6; Upstream and downstream primers and probes for detecting Bacteroides fragilis as shown in SEQ ID NOs: 7 to 9; Upstream and downstream primers and probes for detecting Streptococcus agalactiae as shown in SEQ ID NOs: 10 to 12; Upstream and downstream primers and probes for detecting Streptococcus salivarius as shown in SEQ ID NOs: 13 to 16; and The upstream and downstream primers and probes for detecting Staphylococcus aureus 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 aureus probe is FAM; the fluorescent reporter group of the Staphylococcus capitis probe is HEX; the fluorescent reporter group of the Bacteroides fragilis probe is ROX; the fluorescent reporter group of the Streptococcus agalactiae probe is CY5; the fluorescent reporter group of the Streptococcus salivarius probe is Quasar 705; and the fluorescent reporter group of the Staphylococcus hominis 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 aureus, Staphylococcus capitis, Bacteroides fragilis, Streptococcus agalactiae, Streptococcus salivarius, or Staphylococcus hominis.

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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