Composition, kit and method for detecting urinary tract infection related pathogens and application

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of urinary tract infection related pathogens, and more particularly relates to detection and differentiation of at least four pathogens in staphylococcus saprophyticus, staphylococcus epidermidis, staphylococcus haemolyticus, staphylococcus lugdunensis, staphylococcus farnesae and staphylococcus hominis. According to the combined detection composition provided by the invention, different pathogens are detected by mainly utilizing a multiple fluorescent PCR analysis method and detecting target spots on the different pathogens; therefore, detection and distinguishing of at least four pathogens such as staphylococcus saprophyticus, staphylococcus epidermidis, staphylococcus haemolyticus, staphylococcus lugdunensis, staphylococcus Warner and staphylococcus hominis can be simultaneously realized in a single-tube reaction system, the detection efficiency is improved, and the detection period is shortened. The composition disclosed by the invention is higher in detection sensitivity (up to 200 copies / mL), and also has better specificity and anti-interference performance.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological detection, in particular, to the detection of pathogens related to urinary tract infection, and more particularly, to the detection and identification of Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis. Background Art

[0002] Staphylococcus saprophyticus is a Gram-positive bacterium that is a common pathogen in uncomplicated urinary tract infections (UTIs) in young women. Its clinical features of UTIs share many similarities with those caused by Escherichia coli, but differ in pathogenesis, seasonal variation, and geographic distribution. The incidence of UTIs caused by Staphylococcus saprophyticus is second only to E. coli, especially in women aged 16 to 25, where it causes an infection rate as high as 42%. In addition, Staphylococcus saprophyticus can also cause complications such as acute pyelonephritis, urethritis, epididymitis, and prostatitis.

[0003] Staphylococcus epidermidis is a common commensal bacterium that is commonly found on human skin and mucous membranes. Although it is usually harmless, it can cause urinary tract infections in certain circumstances, such as when the body's immunity is weakened or the bacteria enter an unusual parasitic site. If untreated, urinary tract infections caused by S. epidermidis can lead to serious complications such as pyelonephritis, bacteremia, infective endocarditis, and osteomyelitis. And S. epidermidis may become resistant to multiple antibiotics, which makes treatment more difficult. S. epidermidis is able to form biofilms, which helps the bacteria survive host defenses and antibiotics, increasing the complexity of treatment.

[0004] Staphylococcus haemolyticus is an increasingly common pathogen in hospital settings that can cause a variety of infections, including urinary tract infections. Urinary tract infections are a hazard caused by this bacterium, and symptoms may include a burning sensation in the urethra, frequent urination, urgency, difficulty urinating, and urinary discharge. In severe cases, if left untreated, complications such as kidney scarring and chronic renal failure may result.

[0005] Staphylococcus lugdunensis is a coagulase-negative staphylococcus that can cause a variety of infections, including urinary tract infections (UTIs). The clinical manifestations of S. lugdunensis infection are similar to other UTIs and may include symptoms such as frequent urination, urgency, and pain when urinating. In terms of treatment, S. lugdunensis is usually sensitive to multiple antibiotics, including traditional penicillins and cephalosporins. However, the resistance situation may vary in different regions, so drug sensitivity testing should be performed before treatment to determine the most appropriate antibiotic.

[0006] Staphylococcus warneri is a coagulase-negative staphylococcus that is commonly found on the skin, but in some cases can also cause urinary tract infections (UTIs). Although it accounts for less than 1% of the skin microbiota, S. warneri may cause significant UTIs in immunocompromised individuals or those with other predisposing factors, such as indwelling catheters, stents, or intermittent bladder catheterization, excessive residual urine volume, or obstructive uropathy of any cause.

[0007] Staphylococcus hominis is a coagulase-negative staphylococcus that is commonly found on human skin and generally does not cause disease. However, under certain conditions, such as when the immune system is compromised or when medical devices are implanted, it may cause urinary tract infections, endocarditis, sepsis, etc. Staphylococcus hominis can cause urinary tract infections such as asymptomatic bacteriuria, cystitis, and pyelonephritis, especially in patients with long-term use of catheters or other medical devices.

[0008] These coagulase-negative staphylococci may develop resistance to some commonly used antibiotics. Confirming the pathogen before treatment helps to select the most appropriate antibiotic to ensure effective treatment and reduce the development of drug resistance. Bacterial culture is the gold standard for diagnosing pathogens, but the isolation and culture of pathogens is time-consuming and cumbersome.

[0009] The current detection methods for a single pathogen mainly include direct smear method, microscopic detection, sporulation experiment, flow cytometry detection and molecular biology technology. Among them, molecular biology method has become a hot spot in the research of Staphylococcus aureus detection due to its high sensitivity, rapidity and low cost. However, there are no related reports on the public combination of the above pathogens.

[0010] Therefore, there is a need in the art for a product that can simply and quickly detect and identify the above-mentioned multiple pathogens with high sensitivity and good specificity. Summary of the invention

[0011] In view of this, in a first aspect, the present invention provides a composition for detecting pathogens associated with urinary tract infection, comprising primers and probes shown for at least four pathogens of Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis:

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

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

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

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

[0016] Upstream primers, downstream primers and probes for detecting Staphylococcus warneri as shown in SEQ ID NOs: 13 to 15; and

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

[0018] The present invention provides a combined detection composition, which mainly uses a multiplex fluorescent PCR analysis method to detect different pathogens by detecting targets on different pathogens, thereby simultaneously realizing the detection and identification of at least four pathogens among saprophytic Staphylococcus, epidermidis Staphylococcus, hemolytic Staphylococcus, lugdunensis Staphylococcus, warneria Staphylococcus and human Staphylococcus in a single tube reaction system. The composition of the present invention has a higher detection sensitivity of 200 copies / mL, good specificity, and more accurate detection, providing clinicians with a more sufficient basis for rapid diagnosis and accelerating the implementation of treatment measures for patients.

[0019] In some embodiments, the composition of the present invention may include one or more sets of the above primers and probes. In this article, "set" refers to upstream primers, downstream primers and probes that match each other to detect a target.

[0020] Furthermore, the composition of the present invention can be arbitrarily combined into any combination of detection corresponding to 6 targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe sets corresponding to the targets. These combinations are all included in the present invention.

[0021] For example, it may include all combinations of the above 6 sets of primers and probes, it may include any 5 sets of the above 6 sets of primers and probes, it may include any 4 sets of the above 6 sets of primers and probes, it may include any 3 sets of the above 6 sets of primers and probes, it may include any 2 sets of the above 6 sets of primers and probes, and it may also include any 1 set of the above 6 sets of primers and probes.

[0022] Furthermore, the fluorescent groups of the composite probes are different from each other and do not interfere with each other.

[0023] 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 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.

[0024] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0025] In some specific embodiments, the probes in the compositions of the present invention are labeled with a fluorescent reporter group at the 5' end.

[0026] Further, the fluorescent reporter group of the probe for detecting saprophytic Staphylococcus as shown in SEQ ID NO:3 is ATTO425; the fluorescent reporter group of the probe for detecting Staphylococcus epidermidis as shown in SEQ ID NO:6 is FAM; the fluorescent reporter group of the probe for detecting hemolytic Staphylococcus as shown in SEQ ID NO:9 is HEX; the fluorescent reporter group of the probe for detecting Staphylococcus lugdunensis as shown in SEQ ID NO:12 is ROX; the fluorescent reporter group of the probe for detecting Staphylococcus warneri as shown in SEQ ID NO:15 is CY5; the fluorescent reporter group of the probe for detecting Staphylococcus hominis as shown in SEQ ID NO:18 is Quasar 705.

[0027] In some specific embodiments, the 3' end of the probe further has a fluorescence quenching group, such as MGB, BHQ1 or BHQ2.

[0028] Furthermore, the components of the composition exist in a mixed form.

[0029] In a second aspect, the invention provides a use of the composition of the invention in preparing a kit for the joint detection and differentiation of urinary tract infection-related pathogens, wherein the pathogens are Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis.

[0030] In a third aspect, the present invention provides a kit for the joint detection and differentiation of pathogens associated with urinary tract infection, the kit comprising the composition of the present invention.

[0031] Furthermore, in each reaction system, the concentration of a single primer is 0.01-0.5 μM, and the concentration of a single probe is 0.005-0.2 μM.

[0032] Furthermore, the kit also includes a negative quality control product and a positive quality control product.

[0033] In a specific embodiment, the negative control is physiological saline, but is not limited to physiological saline, and can also be at least one of DEPC H2O and internal standard gene.

[0034] Commonly used internal standards include exogenous non-human genome internal standards, human genome internal standards, artificially synthesized internal standards, etc.

[0035] In a specific embodiment, the positive control is at least one of a plasmid or DNA fragment of Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri, and Staphylococcus hominis.

[0036] Furthermore, the kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a UNG enzyme, a DNA polymerase, dNTPs (U) and Mg 2+ At least one of .

[0037] In some specific embodiments, the DNA polymerase is H-Taq enzyme.

[0038] In a specific embodiment, the kit of the present invention comprises PCR buffer, UNG enzyme, H-Taq enzyme, dNTPs (U), primers, probe and Mg 2+ .

[0039] Common PCR buffers are composed of Tris-HCl, KCl, MgCl2 and other buffer systems. The total volume in a single PCR reaction tube is generally 20 to 200 μL.

[0040] In a fourth aspect, the present invention provides a method for joint detection and differentiation of pathogens associated with urinary tract infection for non-diagnostic purposes, the method comprising the following steps:

[0041] (1) extracting or releasing nucleic acid from the sample to be tested;

[0042] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0043] (3) Obtain and analyze the results.

[0044] In the present invention, the sample used for detection is urine, but is not limited thereto.

[0045] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0046] UNG enzyme reaction, temperature is 50℃, time is 2min, 1 cycle; Taq enzyme activation, temperature is 94℃, time is 3min; denaturation, temperature is 94℃, time is 15s, annealing, temperature is 60℃, time is 15s, extension, temperature is 60℃, time is 15s, 45 cycles, fluorescence collection.

[0047] In a specific embodiment, the present invention provides a composition for preparing a reagent for joint detection and differentiation of urinary tract infection-related pathogens, the use comprising the following steps:

[0048] (1) extracting or releasing nucleic acid from the sample to be tested;

[0049] (2) performing fluorescent quantitative PCR on the nucleic acid obtained in step (1) using the composition of the present invention or the kit of the present invention;

[0050] (3) Obtain and analyze the results.

[0051] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0052] UNG enzyme reaction, temperature is 50℃, time is 2min, 1 cycle; Taq enzyme activation, temperature is 94℃, time is 3min; denaturation, temperature is 94℃, time is 15s, annealing, temperature is 60℃, time is 15s, extension, temperature is 60℃, time is 15s, 45 cycles, fluorescence collection.

[0053] 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 diseases such as urinary tract infection. For example, the presence of the above pathogens can be detected in a test culture (such as urine, etc.).

[0054] In the present invention, the term "primer" refers to two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target gene, and the other primer is complementary to another DNA template strand at the other end of the target gene.

[0055] In the present invention, the term "probe" refers to a nucleic acid sequence (DNA or RNA) with a detection label, a known sequence, and complementary to the target gene.

[0056] In the present invention, the term "nucleotide sequence" refers to the order of bases in DNA or RNA.

[0057] The above scheme of the present invention has the following beneficial effects:

[0058] The present invention is to detect and distinguish urinary tract infection-related pathogens, including the detection and differentiation of at least four pathogens such as saprophytic Staphylococcus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis. The joint inspection composition provided by the present invention mainly 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 four pathogens such as saprophytic Staphylococcus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis in a single tube reaction system, thereby improving the detection efficiency and shortening the detection cycle. The composition of the present invention has a higher detection sensitivity of 200 copies / mL, and also has good specificity and anti-interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0060] Figure 1 It is an amplification curve diagram of the combined detection of the composition of the present invention against Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis;

[0061] Figure 2 It is an amplification curve diagram of the combined detection of the composition of the present invention against Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, and Staphylococcus warneri;

[0062] Figure 3 It is an amplification curve diagram of the combined detection of saprophytic Staphylococcus, epidermidis Staphylococcus, hemolytic Staphylococcus, and lugdunensis by the composition of the present invention;

[0063] Figure 4 is a sensitivity result diagram of the composition of the present invention (Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis);

[0064] Figure 5 is a specific result diagram of the composition of the present invention;

[0065] Figure 6 It is an amplification curve diagram of the comparative example composition of the present invention for joint detection of Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis. DETAILED DESCRIPTION

[0066] The present invention aims at the existing problems and provides a composition, a kit, a method and its use for detecting pathogens related to urinary tract infection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0067] The disclosed embodiments are described to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

[0068] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0069] Example 1: Primers and probes used in the present invention

[0070] The primers and probes used in the present invention are shown in Table 1 below.

[0071] Table 1

[0072]

[0073]

[0074] Among them, the fluorescent reporter group of the probe for detecting saprophytic Staphylococcus as shown in SEQ ID NO:3 is ATTO 425; the fluorescent reporter group of the probe for detecting Staphylococcus epidermidis as shown in SEQ ID NO:6 is FAM; the fluorescent reporter group of the probe for detecting hemolytic Staphylococcus as shown in SEQ ID NO:9 is HEX; the fluorescent reporter group of the probe for detecting Staphylococcus lugdunensis as shown in SEQ ID NO:12 is ROX; the fluorescent reporter group of the probe for detecting Staphylococcus warneri as shown in SEQ ID NO:15 is CY5; the fluorescent reporter group of the probe for detecting Staphylococcus hominis as shown in SEQ ID NO:18 is Quasar 705.

[0075] Example 2: Method for detecting pathogens

[0076] (1) Reagent preparation:

[0077] According to the number of samples to be tested, positive controls, and negative controls, take the corresponding amount of PCR reaction solution and enzyme mixture according to the proportion (PCR reaction solution 38 μL / person + enzyme mixture 2 μL / person), mix them thoroughly to form a PCR mixture, and set aside.

[0078] Sterile saline was used as the negative control matrix, and mixed plasmids containing the specific gene target sequences listed in Table 1 were used as the positive control.

[0079] Preparation of enzyme mixture:

[0080] The enzyme mixture consists of UNG enzyme and H-Taq enzyme. H-Taq enzyme (5U / μL) and UNG enzyme (2U / μL) are mixed in a certain ratio (1.9μL H-Taq enzyme and 0.1μL UNG enzyme per person). The enzyme mixture is used in an amount of 2μL per person.

[0081] (2) Sample processing and sample addition

[0082] Take 300 μL of the sample to be tested, negative control, and positive control respectively into a 1.5 mL centrifuge tube, and use conventional nucleic acid extraction or purification reagents according to their instructions to extract nucleic acid.

[0083] 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.

[0084] The real-time fluorescence PCR reaction system is configured according to Table 2 below:

[0085] Table 2

[0086] Components Volume / concentration in each reaction PCR buffer 34.3μL dNTPs(U)(100mM) 0.5μL <![CDATA[1mol / LMgCl2]]> 0.2μL Primer (50pmol / μL) 0.2μL / strip Probe (50pmol / μL) 0.1μL / strip Sample DNA 10μL

[0087] Note: 0.2 μL / strip means that the volume of each primer is 0.2 μL, and the present invention has 12 primers in total; 0.1 μL / strip means that the volume of each probe is 0.1 μL, and the present invention has 6 probes in total.

[0088] (3) The amplification reaction procedure is shown in Table 3.

[0089] Table 3

[0090]

[0091] (4) Detection

[0092] If the ATTO 425, FAM, HEX, CY5, ROX, and Quasar705 channels have obvious S-shaped amplification curves and the Ct value is ≤38, it is considered positive;

[0093] If there is no amplification curve (NoCt) or Ct value > 38 for ATTO 425, FAM, HEX, ROX and Quasar705 channels, and CY5 ≤ 38, it is considered negative;

[0094] If there is no amplification curve (NoCt) or Ct value>38 for ATTO 425, FAM, HEX, ROX and Quasar705 channels, and CY5>38, the result is unreliable and needs to be retested; as shown in Table 4:

[0095] Table 4

[0096]

[0097] Example 3: Test results of the test samples of the composition of the present invention

[0098] The primers and probes shown in Example 1 were verified on samples of saprophytic Staphylococcus, epidermidis Staphylococcus, hemolytic Staphylococcus, lugdunensis Staphylococcus, Warnera Staphylococcus and human Staphylococcus according to the method of Example 2. The results showed that saprophytic Staphylococcus, epidermidis Staphylococcus, hemolytic Staphylococcus, lugdunensis Staphylococcus, Warnera Staphylococcus and human Staphylococcus could be jointly detected and distinguished.

[0099] Among them, the results of the joint test of Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis are as follows Figure 1 shown.

[0100] The results of the joint test for Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis and Staphylococcus warneri are as follows Figure 2 shown.

[0101] The results of the joint test for Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus lugdunensis are as follows Figure 3 shown.

[0102] from Figures 1 to 3 It can be seen that the composition of the present invention can perform joint detection and differentiation of four, five or six types of Staphylococci.

[0103] Example 4: Sensitivity of the composition of the present invention

[0104] The composition in Example 1 of the present invention was used to perform LOD (sensitivity) detection on various pathogens. The results are shown in Table 5. The detection results show that the composition of the present invention can still accurately detect each channel of samples as low as 200 copies / mL, indicating that the sensitivity of the composition of the present invention is 200 copies / mL. Figure 4 shown.

[0105] Table 5

[0106]

[0107] Example 5. Specificity of the composition of the present invention

[0108] Using the fluorescent PCR reaction system in Example 2 of the present invention, and using the primer probe sequences and fluorescent reporter groups corresponding to Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis in Table 1, according to the detection method in Example 2, detection is performed in reaction systems containing other urinary tract infection pathogens (Staphylococcus aureus, Escherichia coli, Proteus, Neisseria gonorrhoeae, Mycoplasma, Chlamydia, Klebsiella, Citrobacter, Enterococcus faecalis and Pseudomonas aeruginosa, etc.), the composition for detecting urinary tract infection-related pathogens in this embodiment has no cross-reaction with the above-mentioned other urinary tract infection pathogens, and the detection results of other urinary tract infection pathogens are as follows. Figure 5 As shown, the results show that the combination of the present invention for detecting pathogens related to urinary tract infection has no non-specific amplification of other pathogens of urinary tract infection when using the combination of the present invention for detecting pathogens related to urinary tract infection, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis, and the blank specificity of the kit is good. This shows that the combination of the present invention for detecting pathogens related to urinary tract infection has good specificity.

[0109] Example 6: Anti-interference performance of the composition of the present invention

[0110] The method in Example 2 was used for detection, except that different interfering substances (dexamethasone (50 μg / mL), gentamicin (50 μg / mL), vancomycin (50 μg / mL), ceftriaxone hydrochloride (50 μg / mL), ciprofloxacin (50 μg / mL), co-trimoxazole (50 μg / mL), clindamycin (50 μg / mL), heme (10 μg / mL), hemoglobin (10%)) were added to the whole system. The detection results are shown in Table 6.

[0111] As can be seen from Table 6, potential PCR inhibitors / interfering substances have no significant effect on the kit of the present invention. It can be considered that the composition of the present invention has strong anti-interference properties.

[0112] Table 6

[0113]

[0114] Comparative Example 1: Other primers and probes designed by the present invention with poor effects

[0115] 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 excluded 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. It is necessary to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection) and consider the mutual interference between different primers and probes, so the primers and probes need to be carefully designed. Therefore, the inventors designed some other primers and probes.

[0116] This comparative example refers to Example 2 to detect pathogens related to urinary tract infection. The difference between this comparative example and Example 2 is that the primer probe sequence of saprophytic Staphylococcus is different. In addition to the above differences, the other materials and process operations used in this comparative example are strictly consistent with those in Example 2. The primer probe sequence of saprophytic Staphylococcus in this comparative example is shown in Table 7 below.

[0117] The primers and probes (SEQ ID NOs: 1-3) in Example 1 were replaced by the primers and probes (SEQ ID NOs: 19-21) in Comparative Example 1, and the primers and probes (SEQ ID NOs: 4-18) in Example 1 were combined to form a composition (SEQ ID NOs: 4-21) to detect the specificity of Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis. Figure 6 shown.

[0118] Depend on Figure 6 It can be seen that the detection of the composition for detecting urinary tract infection-related pathogens in comparative example 1 (primer probe sequence SEQ ID NO: 4-21) in the six-joint detection system is affected, and the performance of Staphylococcus epidermidis in its amplification curve graph is reduced, the Ct value is delayed, and the fluorescence increment decreases. This fully demonstrates the superiority of the composition of the present invention, and the mutual interference between different primer probes is small. In other words, the combination of primer probes in the present invention is irreplaceable and replaceable.

[0119] Table 7

[0120]

[0121] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition for joint detection and differentiation of pathogens related to urinary tract infection, characterized in that: Includes at least 4 pathogens among Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri, and Staphylococcus hominis. Primers and probes shown: Upstream primers, downstream primers and probes for detecting Staphylococcus saprophyticus as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting Staphylococcus epidermidis as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting hemolytic Staphylococcus as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting Staphylococcus lugdunensis as shown in SEQ ID NOs: 10 to 12; Upstream primers, downstream primers and probes for detecting Staphylococcus warneri as shown in SEQ ID NOs: 13 to 15; as well as The upstream primers, downstream primers and probe for detecting Staphylococcus aureus are shown in SEQ ID NOs: 16-18.

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

3. The composition according to claim 1, characterized in that The fluorescent reporter group of the probe for detecting saprophytic Staphylococcus as shown in SEQ ID NO:3 is ATTO 425; the fluorescent reporter group of the probe for detecting Staphylococcus epidermidis as shown in SEQ ID NO:6 is FAM; the fluorescent reporter group of the probe for detecting hemolytic Staphylococcus as shown in SEQ ID NO:9 is HEX; the fluorescent reporter group of the probe for detecting Staphylococcus lugdunensis as shown in SEQ ID NO:12 is ROX; the fluorescent reporter group of the probe for detecting Staphylococcus warneri as shown in SEQ ID NO:15 is CY5; the fluorescent reporter group of the probe for detecting Staphylococcus hominis as shown in SEQ ID NO:18 is Quasar 705.

4. The composition according to any one of claims 1 to 3, characterized in that The components of the composition are present in admixture.

5. Use of the composition according to any one of claims 1 to 4 in the preparation of a reagent or kit for joint detection and differentiation of pathogens related to urinary tract infection, wherein: The pathogens are Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri and Staphylococcus hominis.

6. A kit for joint detection and differentiation of pathogens related to urinary tract infection, characterized in that: The kit comprises the composition according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that In each reaction system, the concentration of a single primer is 0.01-0.5 μM, and the concentration of a single probe is 0.005-0.2 μM.

8. The kit according to claim 6 or 7, characterized in that The kit also includes a negative quality control product and a positive quality control product.

9. The kit according to any one of claims 6 to 8, characterized in that The kit also includes: a nucleic acid release reagent, a nucleic acid extraction reagent, a PCR buffer, a UNG enzyme, a DNA polymerase, dNTPs (U) and Mg 2+ At least one of .

10. A method for joint detection and differentiation of pathogens associated with urinary tract 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 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 6 to 9; (3) Obtain and analyze the results.

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