Composition and kit for joint detection and differentiation of bloodstream infection pathogens, and use

Abstract

Provided are a composition and kit for joint detection and differentiation of bloodstream infection pathogens, and a use. According to the provided composition for joint detection, by mainly using a multiple fluorescence PCR analysis method, different pathogens are detected by detecting targets on different pathogens, so that the detection and differentiation of Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii are both achieved in a single-tube reaction system.

Description

Composition, kit and use for joint detection and differentiation of bloodstream infection pathogens Technical Field

[0001] The present disclosure belongs to the field of molecular biological detection, and specifically, relates to the detection of pathogens related to bloodstream infection, and more specifically, to the detection of Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii. Background Art

[0002] Burkholderia cepacia is one of several important opportunistic pathogens that cause nosocomial infections. While generally nonpathogenic to healthy individuals, it often invades immunocompromised individuals and frequently colonizes the lungs of patients with cystic fibrosis and chronic granulomatous disease. It is a highly virulent and lethal pathogen. In recent years, the incidence of clinical isolation of this bacterium has increased, and it exhibits high resistance to multiple antibiotics, making its clinical treatment difficult. This aerobic, non-fermenting, Gram-negative, non-spore-forming bacillus is commonly found in moist soil, water, and plants. It can survive and grow in nutrient-deficient water, making it a common pathogen of nosocomial infections. It can be isolated from hospital tap water, thermometers, nebulizers, and urinary catheters, and can also be detected in a variety of clinical samples, including bronchoalveolar lavage fluid, sputum, blood, punctured tissue, and pus. Burkholderia cepacia has unique adhesins that allow it to be transmitted through close contact or inhalation of infected aerosols. It often attacks immunocompromised individuals and is particularly prevalent in intensive care unit (ICU) patients. ICU patients often present with prolonged bed rest, advanced age, compromised immune function, and impaired consciousness. Normal physiological reflexes such as swallowing and coughing are diminished or absent to varying degrees, and sputum, blood, and vomitus are difficult to expel. Repeated invasive respiratory procedures, such as tracheotomy, intubation, and mechanical ventilation, are all risk factors for Burkholderia cepacia infection. Symptoms caused by this infection include bacteremia, urinary tract infections, septic arthritis, endocarditis, meningitis, and respiratory infections.

[0003] Serratia marcescens is a major opportunistic pathogen causing nosocomial infections. The smallest bacteria are approximately 0.5 × (0.5-1.0) micrometers. They are short, nearly spherical rods, but exhibit diverse morphologies. They are Gram-negative, possess flagella throughout their bodies, and are motile. They lack a capsule and spores, and approximately half of their strains produce the red fungus prodigiosin. They are widely distributed in nature, resident in water and soil, and often adhere to ward environments, medical equipment, implanted catheters, and the hands of medical staff. They can also be detected in various clinical samples, including sputum, urine, pus, wound secretions, pleural effusions, bile, drainage fluids, throat swabs, and blood. Serratia marcescens is a common nosocomial infection and can cause sepsis, bacteremia, bloodstream infections, meningitis, endocarditis, myocarditis, pneumonia, cellulitis, wound infections, peritonitis, keratitis, endophthalmitis, urinary tract infections, empyema, cholecystitis, arthritis, osteomyelitis, necrotizing fasciitis, septic shock, suppurative thyroiditis, empyema, epidural abscess, chorioamnionitis, and abscesses. Geriatrics, respiratory medicine, and intensive care units are the primary wards where Serratia marcescens is detected. Its inherent resistance to multiple drugs, such as ampicillin, amoxicillin, macrolides, first-generation cephalosporins, second-generation cephalosporins, and cephamycins, and its tenacity make the infections it causes more frequent and clinical treatment more difficult. In recent years, its resistance to carbapenems (such as meropenem and ertapenem) has also been increasing.

[0004] Acinetobacter baumannii is the most common Gram-negative bacillus in the genus Acinetobacter. It is widely found in water and soil in nature, in hospital environments, and on human skin, as well as in the respiratory, digestive, and genitourinary tracts. It is a conditionally pathogenic bacterium. This bacterium is widely distributed in hospital environments and can survive for long periods of time, making it highly susceptible to infections in critically ill patients. Therefore, it is often isolated from specimens such as blood, urine, pus, and respiratory secretions from infected patients. Among non-fermenting bacteria, its infection rate is second only to Pseudomonas. Infections with Acinetobacter baumannii are gradually increasing, and its increasing drug resistance has drawn significant attention from clinicians and microbiologists. Acinetobacter baumannii primarily causes respiratory infections, but can also lead to sepsis, urinary tract infections, and secondary meningitis. It is widely distributed in hospital environments and can survive for long periods of time, posing a significant threat to critically ill patients and those in CCUs and ICUs. This type of infection is also referred to as an ICU-acquired infection.

[0005] The aforementioned Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii can all cause sepsis or bacteremia, posing a life-threatening threat. Therefore, early diagnosis of the pathogen and prompt, effective anti-infective treatment are key to improving prognosis and significantly reducing morbidity and mortality. However, clinical symptoms and routine laboratory tests are difficult to identify and confirm the infecting bacterial species. Furthermore, current bacterial culture conditions are relatively demanding, resulting in low culture positivity rates and making the culture of multiple mixed infections extremely difficult. This presents significant challenges for infected patients and clinicians.

[0006] Therefore, there is a need in this field for a product that can simply and quickly detect the above pathogens with high sensitivity and good specificity.

[0007] Summary of the Invention

[0008] In view of this, in a first aspect, the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:

[0009] A primer set for detecting Burkholderia cepacia, comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2, a primer pair consisting of an upstream primer as shown in SEQ ID NO:10 and a downstream primer as shown in SEQ ID NO:11, and a primer pair consisting of an upstream primer as shown in SEQ ID NO:13 and a downstream primer as shown in SEQ ID NO:14, and the probe comprises a probe as shown in at least one of the sequences of SEQ ID NO:3, SEQ ID NO:12, and SEQ ID NO:15;

[0010] A primer set for detecting Serratia marcescens, comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer shown in SEQ ID NO:4 and a downstream primer shown in SEQ ID NO:5, a primer pair consisting of an upstream primer shown in SEQ ID NO:16 and a downstream primer shown in SEQ ID NO:17, and a primer pair consisting of an upstream primer shown in SEQ ID NO:19 and a downstream primer shown in SEQ ID NO:20, and the probe comprises a probe represented by at least one of the sequences of SEQ ID NO:6, SEQ ID NO:18, and SEQ ID NO:21; and

[0011] A primer set for detecting Acinetobacter baumannii includes a primer pair and a probe, the primer pair including at least one of a primer pair consisting of an upstream primer shown in SEQ ID NO: 7 and a downstream primer shown in SEQ ID NO: 8, a primer pair consisting of an upstream primer shown in SEQ ID NO: 22 and a downstream primer shown in SEQ ID NO: 23, and a primer pair consisting of an upstream primer shown in SEQ ID NO: 25 and a downstream primer shown in SEQ ID NO: 26, and the probe includes a probe shown in at least one of SEQ ID NO: 9, SEQ ID NO: 24, and SEQ ID NO: 27.

[0012] In some embodiments, the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:

[0013] The upstream primer shown in SEQ ID NO: 1, the downstream primer shown in SEQ ID NO: 2, and the probe shown in SEQ ID NO: 3 are used to detect Burkholderia cepacia;

[0014] The upstream primer shown in SEQ ID NO: 4, the downstream primer shown in SEQ ID NO: 5, and the probe shown in SEQ ID NO: 6 are used to detect Serratia marcescens; and

[0015] The upstream primer shown in SEQ ID NO: 7, the downstream primer shown in SEQ ID NO: 8, and the probe shown in SEQ ID NO: 9 are used to detect Acinetobacter baumannii.

[0016] In a second aspect, the present disclosure provides a use of the above-mentioned composition in preparing a kit for joint detection and differentiation of bloodstream infection pathogens, wherein the pathogens are Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii.

[0017] In a third aspect, the present disclosure provides a kit for the joint detection and differentiation of bloodstream infection pathogens, the kit comprising the composition as described above.

[0018] In a fourth aspect, the present disclosure provides a use for preparing a reagent for joint detection and differentiation of bloodstream infection pathogens, wherein the joint detection comprises the following steps:

[0019] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0020] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present disclosure or the kit of the present disclosure as described above;

[0021] 3) Obtain and analyze the results.

[0022] The combined detection composition provided by the present disclosure primarily utilizes a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously detecting and differentiating Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii in a single-tube reaction system. The disclosed composition has a higher sensitivity of up to 500 copies / mL, good specificity, and more accurate detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1a is a graph showing the detection results of a composition disclosed herein.

[0024] FIG1b is a graph showing the test results of a composition disclosed herein.

[0025] FIG1c is a graph showing the detection results of a composition disclosed herein.

[0026] FIG2 is a graph showing the sensitivity results of a composition disclosed herein for detecting Burkholderia cepacia.

[0027] FIG3 is a graph showing the sensitivity results of a composition disclosed herein for detecting Acinetobacter baumannii.

[0028] FIG4 is a graph showing the sensitivity results of a composition disclosed herein for detecting Serratia marcescens.

[0029] FIG5 is a graph showing the specificity results of the disclosed composition.

[0030] FIG6 shows the results of gene amplification detection of the selected Burkholderia cepacia scpA-1 and Acinetobacter baumannii rlmA-1 / rlmA-2 combination genes.

[0031] FIG7 shows the gene amplification detection results of the selected Burkholderia cepacia scpA-1 and Serratia marcescens M60-1 / M60-2 combination.

[0032] FIG8 shows the gene amplification detection results of the selected Serratia marcescens M60-1 and Burkholderia cepacia scpA-1 / scpA-2 combination.

[0033] FIG9 is a graph showing the detection results of the single target primer and the comparative primer of the composition disclosed herein for detecting Burkholderia cepacia.

[0034] FIG10 is a graph showing the detection results of the single target primer and the comparative primer of the composition for detecting Acinetobacter baumannii disclosed herein.

[0035] FIG11 is a graph showing the detection results of the single target primer and the comparative primer of the composition for detecting Serratia marcescens disclosed herein. DETAILED DESCRIPTION

[0036] The present disclosure will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present disclosure 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 disclosure, rather than to limit the present disclosure.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents, materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0038] In a first aspect, the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:

[0039] A primer set for detecting Burkholderia cepacia, comprising a primer pair and a probe, wherein the primer pair comprises at least one of an upstream primer as shown in SEQ ID NO: 1 and a downstream primer as shown in SEQ ID NO: 2, a primer pair as shown in SEQ ID NO: 10 and a downstream primer as shown in SEQ ID NO: 11, and a primer pair as shown in SEQ ID NO: 13 and a downstream primer as shown in SEQ ID NO: 14, and the probe comprises a probe as shown in at least one of the sequences of SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 15;

[0040] A primer set for detecting Serratia marcescens, comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer shown in SEQ ID NO:4 and a downstream primer shown in SEQ ID NO:5, a primer pair consisting of an upstream primer shown in SEQ ID NO:16 and a downstream primer shown in SEQ ID NO:17, and a primer pair consisting of an upstream primer shown in SEQ ID NO:19 and a downstream primer shown in SEQ ID NO:20, and the probe comprises a probe represented by at least one of the sequences of SEQ ID NO:6, SEQ ID NO:18, and SEQ ID NO:21; and

[0041] A primer set for detecting Acinetobacter baumannii includes a primer pair and a probe, the primer pair including at least one of a primer pair consisting of an upstream primer shown in SEQ ID NO: 7 and a downstream primer shown in SEQ ID NO: 8, a primer pair consisting of an upstream primer shown in SEQ ID NO: 22 and a downstream primer shown in SEQ ID NO: 23, and a primer pair consisting of an upstream primer shown in SEQ ID NO: 25 and a downstream primer shown in SEQ ID NO: 26, and the probe includes a probe shown in at least one of SEQ ID NO: 9, SEQ ID NO: 24, and SEQ ID NO: 27.

[0042] In some embodiments, the primer set for detecting Burkholderia cepacia is selected from at least one of the following primer sets: a primer set consisting of an upstream primer as shown in SEQ ID NO: 1, a downstream primer as shown in SEQ ID NO: 2, and a probe as shown in SEQ ID NO: 3, a primer set consisting of an upstream primer as shown in SEQ ID NO: 10, a downstream primer as shown in SEQ ID NO: 11, and a probe as shown in SEQ ID NO: 12, or a primer set consisting of an upstream primer as shown in SEQ ID NO: 13, a downstream primer as shown in SEQ ID NO: 14, and a probe as shown in SEQ ID NO: 15.

[0043] In some embodiments, the primer set for detecting Serratia marcescens is selected from at least one of the following primer sets: a primer set consisting of an upstream primer shown in SEQ ID NO:4, a downstream primer shown in SEQ ID NO:5, and a probe shown in SEQ ID NO:6, a primer set consisting of an upstream primer shown in SEQ ID NO:16, a downstream primer shown in SEQ ID NO:17, and a probe shown in SEQ ID NO:18, or a primer set consisting of an upstream primer shown in SEQ ID NO:19, a downstream primer shown in SEQ ID NO:20, and a probe shown in SEQ ID NO:21.

[0044] In some embodiments, the primer set for detecting Acinetobacter baumannii is selected from at least one of the following primer sets: a primer set consisting of an upstream primer as shown in SEQ ID NO: 7, a downstream primer as shown in SEQ ID NO: 8, and a probe as shown in SEQ ID NO: 9, a primer set consisting of an upstream primer as shown in SEQ ID NO: 22, a downstream primer as shown in SEQ ID NO: 23, and a probe as shown in SEQ ID NO: 24, or a primer set consisting of an upstream primer as shown in SEQ ID NO: 25, a downstream primer as shown in SEQ ID NO: 26, and a probe as shown in SEQ ID NO: 27.

[0045] In some embodiments, the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:

[0046] Upstream primers, downstream primers, and probes for detecting Burkholderia cepacia as shown in SEQ ID NOs: 1 to 3;

[0047] Upstream primers, downstream primers, and probes for detecting Serratia marcescens as shown in SEQ ID NOs: 4 to 6; and

[0048] The upstream primers, downstream primers and probes for detecting Acinetobacter baumannii are shown in SEQ ID NOs: 7 to 9.

[0049] In some embodiments, the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:

[0050] The upstream primer shown in SEQ ID NO: 1, the downstream primer shown in SEQ ID NO: 2, and the probe shown in SEQ ID NO: 3 are used to detect Burkholderia cepacia;

[0051] The upstream primer shown in SEQ ID NO: 4, the downstream primer shown in SEQ ID NO: 5, and the probe shown in SEQ ID NO: 6 are used to detect Serratia marcescens; and

[0052] The upstream primer shown in SEQ ID NO: 7, the downstream primer shown in SEQ ID NO: 8, and the probe shown in SEQ ID NO: 9 are used to detect Acinetobacter baumannii.

[0053] In some embodiments, the present disclosure provides a composition for joint detection and differentiation of bloodstream infection pathogens, comprising:

[0054] The upstream primer shown in SEQ ID NO: 1, the downstream primer shown in SEQ ID NO: 2, and the probe shown in SEQ ID NO: 3 are used to detect Burkholderia cepacia;

[0055] The upstream primer shown in SEQ ID NO: 4, the downstream primer shown in SEQ ID NO: 5, and the probe shown in SEQ ID NO: 6 are used to detect Serratia marcescens; and

[0056] The upstream primer shown in SEQ ID NO: 7, the downstream primer shown in SEQ ID NO: 8, and the probe shown in SEQ ID NO: 9 are used to detect Acinetobacter baumannii.

[0057] The combined detection composition provided herein primarily utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously detecting and differentiating Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii in a single-tube reaction system. The disclosed composition has a higher sensitivity of up to 500 copies / mL, good specificity, and more accurate detection.

[0058] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0059] In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is RNase P.

[0060] In some embodiments, the probes in the composition are labeled with a fluorescent reporter group at the 5' end.

[0061] Furthermore, the fluorescent reporter groups of the probes of the disclosed composition are different from each other and do not interfere with each other.

[0062] In this context, "different and non-interfering" means that the fluorescent reporter groups used in each probe in the composition are different and do not affect each other's detection, that is, they can be detected using different channels. For example, ATTO 425, Quasar 705, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values ​​and can be used in different channels, thus preventing interference.

[0063] In some specific embodiments, the fluorescent reporter group of the Burkholderia cepacia probe is FAM; the fluorescent reporter group of the Acinetobacter baumannii probe is HEX (or VIC); and the fluorescent reporter group of the Serratia marcescens probe is ROX.

[0064] Furthermore, in some embodiments, the compositions of the present disclosure may include one or more primer sets consisting of the above primer pairs and probes. In the present disclosure, a "primer set" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0065] The compositions disclosed herein can be combined in any combination to detect corresponding targets. Those skilled in the art can combine them as needed to determine which targets to detect, i.e., by combining primer and probe pairs corresponding to the targets. These combinations are encompassed by the present disclosure.

[0066] In some specific embodiments, the disclosed compositions are used for fluorescent PCR.

[0067] Furthermore, the 3' end of the probe also has a non-fluorescent quencher, such as NFQ and QSY.

[0068] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0069] In a specific embodiment, the 3' end of the probe is BHQ1.

[0070] In a specific embodiment, each component of the composition of the present disclosure is present in a separate package.

[0071] In a particular embodiment, the components of the composition of the present disclosure are present in the same package.

[0072] Furthermore, the components of the composition of the present disclosure are present in a mixed form.

[0073] In a second aspect, the present disclosure provides a use of the above-mentioned composition of the present disclosure in preparing a kit for joint detection and differentiation of bloodstream infection pathogens, wherein the pathogens are Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii.

[0074] In a third aspect, the present disclosure provides a kit for the joint detection and differentiation of bloodstream infection pathogens, wherein the kit comprises the composition of the present disclosure as described above.

[0075] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0076] In a specific embodiment, the negative control is at least one of DEPC H2O, physiological saline, and an internal standard gene. The positive control is at least one of a plasmid fragment or a DNA fragment of Burkholderia cepacia, Serratia marcescens, or Acinetobacter baumannii.

[0077] Furthermore, the kit also includes dNTP, PCR buffer and Mg 2+At least one of .

[0078] Furthermore, the kit further comprises: at least one of a nucleic acid releasing reagent, a nucleic acid extracting reagent, and a DNA polymerase.

[0079] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UDG), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0080] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction. For example, the DNA polymerase may be Taq enzyme.

[0081] In a specific embodiment, the kit of the present disclosure includes Taq enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0082] Common PCR buffers are composed of Tris-HCl, MgCl2, KCl, Triton X-100, etc. The total volume of a single PCR reaction tube is generally 20μl to 200μl.

[0083] In a specific embodiment, the kit disclosed herein is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0084] In a fourth aspect, a method for preparing a reagent for joint detection and differentiation of bloodstream infection pathogens is provided, wherein the joint detection comprises the following steps:

[0085] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0086] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present disclosure or the kit of the present disclosure as described above;

[0087] 3) Obtain and analyze the results.

[0088] The present disclosure also provides a method for joint detection and differentiation of bloodstream infection pathogens, the method comprising the following steps:

[0089] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0090] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present disclosure or the kit of the present disclosure as described above;

[0091] 3) Obtain and analyze the results.

[0092] In one embodiment, the method can be used for the diagnosis and treatment of a disease, can be used for auxiliary diagnosis of a disease, or can be used for neither diagnosis nor treatment of a disease.

[0093] In one embodiment, the method is not intended for diagnosis of a disease, i.e., for non-diagnostic purposes. As used herein, the term "non-diagnostic purpose" refers to a method not intended to obtain information about whether an individual is infected with the aforementioned pathogen and has suffered a bloodstream infection. For example, the method may be used to detect the presence of the aforementioned pathogen in a test culture (e.g., blood).

[0094] In one embodiment, a method for joint detection and differentiation of bloodstream infection pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

[0095] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0096] 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of the present disclosure or the kit of the present disclosure as described above;

[0097] 3) Obtain and analyze the results.

[0098] In the present disclosure, the sample used for detection may be blood, plasma, etc., but is not limited thereto. It may also be alveolar lavage fluid, sputum, punctured tissue, pus, wound secretions, etc.

[0099] Furthermore, the reaction conditions of the fluorescent quantitative PCR are as follows: DNA pre-denaturation at 95°C for 1 to 3 minutes, 1 cycle; denaturation at 95°C for 5 to 20 seconds, annealing at 55°C to 60°C for 10 to 60 seconds, 30 to 50 cycles, and fluorescence collection.

[0100] Example 1: Primers and probes used in this disclosure

[0101] The primers and probes used in the present disclosure are shown in Table 1. Sequences with a similarity of 80% or more to the sequences (SEQ ID NO: 1 to SEQ ID NO: 9) can be amplified.

[0102] Table 1

[0103] Among them, the fluorescent reporter group of the Burkholderia cepacia scpA probe is FAM; the fluorescent reporter group of the Serratia marcescens M60 probe is ROX; and the fluorescent reporter group of the Acinetobacter baumannii rlmA probe is HEX.

[0104] Example 2: Method for detecting pathogens

[0105] Fluorescence PCR amplification reaction solution: contains PCR buffer, Taq enzyme, Mg 2+ , dNTPs, primers, probes, etc. The specific reaction system is shown in Table 2.

[0106] Table 2

[0107] The amplification reaction procedure is shown in Table 3.

[0108] Table 3

[0109] Result analysis and judgment:

[0110] After the reaction is complete, the results are automatically saved and the amplification curves of the detected targets are analyzed separately. Based on the analyzed image, adjust the Baseline Start, End, and Threshold values ​​(users can adjust the Start value to 3-15 and the End value to 5-20 based on actual conditions. Adjust the negative control amplification curve to make it flat or below the threshold line). Click Analyze to ensure that all parameters meet the requirements of "Quality Control" below. Then, record the qualitative results in the Plate window.

[0111] Quality Control

[0112] Negative control: No Ct value is displayed in the curves of the three channels of FAM, ROX, and HEX;

[0113] Positive control: Ct of the curves of the three channels of FAM, ROX, and HEX is ≤ 36;

[0114] The above requirements must be met simultaneously in the same experiment; otherwise, the experiment will be invalid and must be repeated.

[0115] Positive judgment value

[0116] The Ct reference value of the target gene detected by this kit was determined to be 38 through reference value research.

[0117] According to the above test results, the judgment results are as follows (see Table 4).

[0118] Table 4

[0119] Example 3. Test results of the test samples of the disclosed composition

[0120] The primers and probes shown in Example 1 were used to perform PCR detection on Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii according to the method of Example 2 on a Macrostone fluorescent quantitative PCR instrument. The test results are shown in Figures 1a to 1c. It can be seen from the figures that the compositions disclosed herein can perform good detection of various pathogens. Although the fluorescence intensity of the compositions provided by SEQ ID NO: 10 to SEQ ID NO: 27 is relatively weak, they can also perform good discrimination of various pathogens.

[0121] Example 4: Sensitivity of the disclosed composition

[0122] Using the composition in Example 1 of the present disclosure (scpA group corresponding to SEQ ID NO: 1 to SEQ ID NO: 3, rlmA group corresponding to SEQ ID NO: 6 to SEQ ID NO: 9, and M60 group corresponding to SEQ ID NO: 4 to SEQ ID NO: 6), LOD (sensitivity) detection was performed on each target (corresponding to Burkholderia cepacia scpA, Acinetobacter baumannii rlmA, and Serratia marcescens M60, respectively) to simulate clinical samples and perform multiplex PCR detection on a macrostone fluorescent quantitative PCR instrument. The results are shown in Figures 2 to 4, indicating that each channel can still accurately detect samples as low as 500 copies / mL, indicating that the sensitivity of the disclosed composition is high, and the detection limit is ≤500 copies / mL.

[0123] Example 5. Specificity of the disclosed composition

[0124] To test the blank specificity of the composition in Example 1 of the present disclosure (the scpA group corresponding to SEQ ID NO: 1 to SEQ ID NO: 3, the rlmA group corresponding to SEQ ID NO: 6 to SEQ ID NO: 9, and the M60 group corresponding to SEQ ID NO: 4 to SEQ ID NO: 6), the negative control was used as a sample and the above-described operating procedures were followed. The results, as shown in Figure 5, showed that there was no nonspecific amplification in each target channel, indicating that the blank specificity of the kit was good.

[0125] Comparative Example 1: Other primers and probes designed by the present disclosure that are not effective

[0126] Due to the principle of complementary base pairing, dimers can form between primers and / or probes, but this probability is very low and can be eliminated at the beginning of the design. However, when testing for multiple pathogens together, there are many primers and probes, and dimers are likely to form between primers, probes, or even between primers. To ensure the conservative 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 must be carefully designed.

[0127] Therefore, the inventors also designed other primers and probes to form different detection systems 1 to 3 (sequences not shown), which were also used to detect the above pathogens. The specific detection results are shown in Figures 6 to 8.

[0128] In addition, the inventors also compared the amplification effects for each target, and the results are shown in Figures 9 to 11. As can be seen from Figures 9 to 11, the remaining primers and probes have a smaller gap in the detection effect of a single target, but in the system of the joint inspection, the gap in its amplification effect is significantly larger than that of the single target detection, which may be due to the influence of the primers and probes on each other in the joint inspection system; specifically, as shown in Figure 9, the primer groups scpA-1 and scpA-2 designed for Burkholderia cepacia respectively have a close detection effect, but Burkholderia cepacia scpA-1 and scpA-2 are mixed with Serratia marcescens primer group M60-1 for amplification detection, and the combination of test results M60-1 and scpA-1 is significantly better than M60-1 and scpA-2. Therefore, the joint inspection system disclosed herein is superior.

[0129] The above describes the embodiments of the present disclosure. However, the present disclosure is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.

Claims

1. A composition for joint detection and differentiation of bloodstream infection pathogens, comprising: A primer set for detecting Burkholderia cepacia, comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO:1 and a downstream primer as shown in SEQ ID NO:2, a primer pair consisting of an upstream primer as shown in SEQ ID NO:10 and a downstream primer as shown in SEQ ID NO:11, and a primer pair consisting of an upstream primer as shown in SEQ ID NO:13 and a downstream primer as shown in SEQ ID NO:14, and the probe comprises a probe as shown in at least one sequence of SEQ ID NO:3, SEQ ID NO:12, and SEQ ID NO:15; A primer set for detecting Serratia marcescens, comprising a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:5, a primer pair consisting of an upstream primer as shown in SEQ ID NO:16 and a downstream primer as shown in SEQ ID NO:17, and a primer pair consisting of an upstream primer as shown in SEQ ID NO:19 and a downstream primer as shown in SEQ ID NO:20, and the probe comprises a probe as shown in at least one sequence among SEQ ID NO:6, SEQ ID NO:18 and SEQ ID NO:21; and A primer set for detecting Acinetobacter baumannii comprises a primer pair and a probe, wherein the primer pair comprises at least one of a primer pair consisting of an upstream primer shown in SEQ ID NO:7 and a downstream primer shown in SEQ ID NO:8, a primer pair consisting of an upstream primer shown in SEQ ID NO:22 and a downstream primer shown in SEQ ID NO:23, and a primer pair consisting of an upstream primer shown in SEQ ID NO:25 and a downstream primer shown in SEQ ID NO:26, and the probe comprises a probe as shown in at least one of SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:

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2. The composition according to claim 1, characterized in that The primer set for detecting Burkholderia cepacia is selected from at least one of the following primer sets: a primer set consisting of an upstream primer as shown in SEQ ID NO: 1, a downstream primer as shown in SEQ ID NO: 2, and a probe as shown in SEQ ID NO: 3, a primer set consisting of an upstream primer as shown in SEQ ID NO: 10, a downstream primer as shown in SEQ ID NO: 11, and a probe as shown in SEQ ID NO: 12, or a primer set consisting of an upstream primer as shown in SEQ ID NO: 13, a downstream primer as shown in SEQ ID NO: 14, and a probe as shown in SEQ ID NO: 15; and / or The primer set for detecting Serratia marcescens is selected from at least one of the following primer sets: a primer set consisting of an upstream primer as shown in SEQ ID NO:4, a downstream primer as shown in SEQ ID NO:5, and a probe as shown in SEQ ID NO:6, a primer set consisting of an upstream primer as shown in SEQ ID NO:16, a downstream primer as shown in SEQ ID NO:17, and a probe as shown in SEQ ID NO:18, or a primer set consisting of an upstream primer as shown in SEQ ID NO:19, a downstream primer as shown in SEQ ID NO:20, and a probe as shown in SEQ ID NO:21; and / or The primer set for detecting Acinetobacter baumannii is selected from at least one of the primer sets shown below: a primer set consisting of an upstream primer as shown in SEQ ID NO:7, a downstream primer as shown in SEQ ID NO:8, and a probe as shown in SEQ ID NO:9, a primer set consisting of an upstream primer as shown in SEQ ID NO:22, a downstream primer as shown in SEQ ID NO:23, and a probe as shown in SEQ ID NO:24, or a primer set consisting of an upstream primer as shown in SEQ ID NO:25, a downstream primer as shown in SEQ ID NO:26, and a probe as shown in SEQ ID NO:

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3. A composition for joint detection and differentiation of bloodstream infection pathogens, comprising: An upstream primer as shown in SEQ ID NO:1, a downstream primer as shown in SEQ ID NO:2, and a probe as shown in SEQ ID NO:3 are used to detect Burkholderia cepacia; The upstream primer as shown in SEQ ID NO:4, the downstream primer as shown in SEQ ID NO:5 and the probe as shown in SEQ ID NO:6 are used to detect Serratia marcescens; as well as The upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8 and the probe shown in SEQ ID NO:9 are used to detect Acinetobacter baumannii.

4. The composition according to any one of claims 1 to 3, 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, and are preferably independently selected from ATTO 425, Quasar705, FAM, HEX, ROX and CY5; More preferably, the fluorescent reporter group of Burkholderia cepacia is FAM; the fluorescent reporter group of Serratia marcescens is HEX or IVC; and the fluorescent reporter group of Acinetobacter baumannii is ROX.

5. The composition according to any one of claims 1 to 4, characterized in that The composition also includes an upstream primer, a downstream primer and a probe for detecting an internal standard; Preferably, the internal standard is a human internal standard gene; More preferably, the internal standard is RNase P.

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. The composition according to any one of claims 1 to 6, characterized in that In the composition, the 3' end of the probe has a non-fluorescent quencher.

8. The composition according to any one of claims 1 to 7, characterized in that In the composition, the 3' end of the probe further has a quencher group.

9. The composition according to claim 8, characterized in that The quenching group is BHQ1 or BHQ2.

10. Use of the composition according to any one of claims 1 to 9 in preparing a kit for joint detection and differentiation of bloodstream infection pathogens, wherein: The pathogens are Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii.

11. A kit for joint detection and differentiation of bloodstream infection pathogens, comprising the composition according to any one of claims 1 to 10.

12. The kit according to claim 11, characterized in that The kit also includes a negative quality control product and a positive quality control product.

13. The kit according to claim 12, characterized in that Negative control is at least one of DEPC H2O, saline, and internal standard gene; and / or The positive quality control product is at least one of the fragment plasmid or fragment DNA of Burkholderia cepacia, Serratia marcescens, and Acinetobacter baumannii.

14. The kit according to any one of claims 11 to 13, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a DNA polymerase, dNTP, dUTP, UDG enzyme, a PCR buffer, and Mg 2+ At least one of .

15. A method for preparing a reagent for joint detection and differentiation of bloodstream infection pathogens, the joint detection comprising the following steps: 1) Extracting or releasing nucleic acid from the sample to be tested; 2) performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 7 or the kit according to any one of claims 11 to 14; 3) Obtain and analyze the results.

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