Compositions, kits and uses for simultaneous detection and differentiation of blood stream infection pathogens

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to detection of blood stream infection related pathogenic bacteria, and more particularly to detection of angina streptococcus, viridans streptococcus and enterococcus faecium. The combined composition provided by the present application mainly utilizes a multiple fluorescence PCR analysis method, and different pathogenic bacteria are detected by detecting target points on different pathogenic bacteria, so that detection and differentiation of angina streptococcus, viridans streptococcus and enterococcus faecium are simultaneously realized in a single tube reaction system, so as to improve the treatment strategy in a targeted manner. The composition of the present application has higher detection sensitivity, reaches 500 copies / mL, has good specificity, and is more accurate in detection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically, it relates to the detection of bloodstream infection-related pathogens, and more specifically, it relates to the detection of Streptococcus pharyngitis, Streptococcus pyogenes, and Enterococcus faecalis. Background Technology

[0002] *Streptococcus anginosus* is a normal flora that colonizes the oral cavity, pharynx, and digestive tract, and is part of the Miller streptococcus group. It is a Gram-positive, catalase-negative facultative anaerobic coccus that forms small colonies on agar media and belongs to the viridans streptococcus group. The most common diseases caused by *Streptococcus anginosus* are respiratory tract infections, purulent infections, and bloodstream infections, accounting for 34.8%, 33.5%, and 8.5% respectively. Previous studies have confirmed that *Streptococcus anginosus* can produce various exotoxins, often causing invasive purulent infections. Once it invades the bloodstream, it can circulate throughout the body, causing serious systemic infectious diseases. Most patients with bloodstream infections caused by *Streptococcus anginosus* are related to invasive procedures. Clinically, most patients with bloodstream infections of *Streptococcus anginosus* have underlying diseases such as digestive, cardiovascular, and respiratory diseases, as well as tumors. Multiple studies have shown that the most common underlying disease in bloodstream infections of Streptococcus pharyngitis is tumors. It is speculated that space-occupying lesions caused by tumors disrupt normal bodily structures, leading to local infection and subsequently Streptococcus pharyngitis. Streptococcus pharyngitis infections are characterized by indistinct features, making identification difficult. In clinical practice, if a related bacterial infection is suspected, specimens should be submitted for testing promptly, and antibiotics should be administered rationally based on drug sensitivity results. Drug resistance monitoring should be conducted to improve prognosis. Regarding the identification of Streptococcus pharyngitis, most countries use automated identification instruments and the API 20Strep reagent kit. Only a few large hospitals in China use these methods, and even with these methods, 100% accurate identification is not guaranteed, but they remain the primary identification methods in clinical laboratories.

[0003] Streptococcus mitis (S. mitis) includes Streptococcus mitis, Streptococcus oralis, Streptococcus infantis, pan-oral Streptococcus, and Streptococcus sternae. Streptococcus mitis is a type of viridans streptococcus and is part of the normal human flora, mainly distributed in the oropharynx, but also found on the skin, in the gastrointestinal tract, and in the female reproductive tract. Streptococcus mitis is a Gram-positive coccus, spherical or oval in shape, forming short or long chains in serum broth. It is non-spore-forming, non-motile, facultatively anaerobic, and catalase-negative. Streptococcus mitis is an opportunistic pathogen that can cause a range of invasive diseases, especially in patients with malignant tumors, undergoing chemotherapy, or those with immunodeficiency such as neutropenia. Streptococcus mitis can cause bloodstream infections, infective endocarditis, meningitis, pneumonia, odontogenic infections, sinusitis, and abscessive lesions. Streptococcus pyogenes possesses the same gene structure encoding gentamicin resistance as Enterococcus faecalis and Enterococcus faecium; this gene determinant is integrated into the chromosome rather than the plasmid. Infections with Streptococcus pyogenes are not uncommon, with various causes and clinical types, but are often missed due to misdiagnosis as viridans streptococci during culture. However, Streptococcus pyogenes develops resistance more rapidly than viridans streptococci; therefore, microbiology labs and clinicians should be more vigilant about infections caused by this bacterium and improve diagnostic accuracy.

[0004] Enterococcus faecium, belonging to the genus Enterococcus, is part of the normal flora of the human and animal intestines. Normally, Enterococcus faecium is present in the intestines of humans and animals and does not cause disease in the host. However, when the host's immunity is weakened or nutritional deficiencies occur, it can lead to pathological changes, typically causing wound infections and peritonitis, and triggering bacteremia. In cases of weakened immunity, excessive use of antibiotics, and mixed infections with multiple pathogens, it can cause sepsis, endocarditis, encephalitis, etc., with a mortality rate reaching 21.0%–27.5%. As an opportunistic pathogen, the infection rate of Enterococcus faecium has been rising in recent years in the United States, Europe, and South America. The China Antimicrobial Resistance Surveillance Network shows that the increasing detection of Enterococcus species year by year is dominated by Enterococcus faecium. Preliminary identification of pathogens using staining morphology and biochemical properties of Enterococcus faecalis is not only time-consuming but also easily confused with other bacterial genera. Therefore, nucleic acid diagnostic methods such as DNA molecular hybridization or polymerase chain reaction (PCR) technology can rapidly diagnose Enterococcus faecalis, which is of great guiding significance for the early diagnosis of clinical infections.

[0005] The aforementioned Streptococcus pharyngitis, Streptococcus suis, and Enterococcus faecalis can all cause sepsis, endangering life. Therefore, early diagnosis of the pathogen and timely and effective anti-infective treatment are crucial for improving prognosis and can significantly reduce morbidity and mortality. However, it is difficult to identify the specific bacteria causing infection through clinical symptoms and routine laboratory tests. Moreover, current bacterial culture conditions are quite stringent, resulting in low positive culture rates, and cultures for multiple mixed infections are extremely difficult. This poses a significant challenge for both infected patients and clinicians.

[0006] Therefore, there is a need in the field for a product that can easily and quickly detect the above-mentioned pathogens in order to improve treatment strategies in a targeted manner, and that has high sensitivity and specificity. Summary of the Invention

[0007] In view of this, in a first aspect, the present invention provides a composition for the joint detection and differentiation of bloodstream infection pathogens, comprising:

[0008] For example, the upstream primer, downstream primer and probe for detecting Streptococcus pharyngitis shown in SEQ ID NO:1-3;

[0009] For example, the upstream primer, downstream primer and probe for detecting Streptococcus pyogenes shown in SEQ ID NO:4-6; and the upstream primer, downstream primer and probe for detecting Enterococcus faecalis shown in SEQ ID NO:7-9.

[0010] The combined detection composition provided by this invention mainly utilizes multiplex fluorescent PCR analysis to detect different pathogens by detecting target sites on different pathogens. This allows for the simultaneous detection and differentiation of *Streptococcus pharyngitis*, *Streptococcus suis*, and *Enterococcus faecalis* in a single-tube reaction system, thereby improving targeted treatment strategies. The composition of this invention exhibits higher detection sensitivity (up to 500 copies / mL), better specificity, and more accurate detection.

[0011] Furthermore, the composition includes an upstream primer, a downstream primer, and a probe for detecting an internal standard.

[0012] In some specific implementations, the internal control is a human internal control gene. In one specific implementation, the internal control is RNase P.

[0013] Furthermore, the fluorescent groups of the probes in the composition of the present invention are different from each other and do not interfere with each other.

[0014] In this article, "dissimilar and non-interfering" means that each probe in the composition uses a different fluorophore and will not affect the detection of each other, i.e., different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX, and CY5 can be used. These groups have different absorbance values ​​and can be selected in different channels, thus avoiding mutual interference.

[0015] In some specific implementations, the fluorescent reporter group of the Streptococcus pharyngitis probe is FAM; the fluorescent reporter group of the Streptococcus suis probe is HEX; and the fluorescent reporter group of the Enterococcus faecium probe is ROX.

[0016] Furthermore, in some embodiments, the composition of the present invention may simultaneously include one or more pairs of the primer and probe pairs described above. In the present invention, a "pair" refers to a mutually matched upstream and downstream primer and probe for detecting a target.

[0017] The compositions of this invention can be arbitrarily combined to detect any combination of three corresponding targets. Those skilled in the art can combine them as needed, determining which targets to detect by combining the primer and probe pairs corresponding to those targets. All such combinations are included in this invention.

[0018] For example, it may include any 3 pairs of the above 3 pairs of primers and probes, any 2 pairs of the above 3 pairs of primers and probes, or any 1 pair of the above 3 pairs of primers and probes.

[0019] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0020] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0021] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.

[0022] In one specific implementation, the 3' end of the probe is BHQ1.

[0023] In one specific embodiment, each component of the composition of the present invention is contained in a separate package.

[0024] In one specific embodiment, the components of the composition of the present invention are contained in the same package.

[0025] Furthermore, the components of the composition of the present invention exist in a mixed form.

[0026] Secondly, the present invention provides the use of the above-described composition of the present invention in the preparation of a kit for the joint detection and differentiation of bloodstream infection pathogens, wherein the pathogens are Streptococcus pharyngitis, Streptococcus pyogenes, and Enterococcus faecalis.

[0027] Thirdly, the present invention provides a kit for the joint detection and differentiation of bloodstream infection pathogens, the kit comprising the composition of the present invention as described above.

[0028] Furthermore, the kit also includes negative and positive controls.

[0029] In one specific implementation, the negative control is at least one of DEPC H2O, physiological saline, and internal standard gene. The positive control is at least one of a fragment plasmid or fragment DNA of Streptococcus pharyngitis, Streptococcus suis, or Enterococcus faecalis.

[0030] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0031] Furthermore, the kit also includes at least one of the following: nucleic acid extraction reagents and DNA polymerase.

[0032] Furthermore, the kit also includes nucleic acid extraction reagents, dNTPs, dUTPs, uracil glycosylase (UDG), DNA polymerase, PCR buffer, and Mg... 2+ At least one of them.

[0033] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase can be Taq polymerase.

[0034] In one specific embodiment, the kit of the present invention includes Taq enzyme, Mg 2+ dNTPs (U), primers, probes, and PCR buffer.

[0035] Common PCR buffers consist of buffer systems such as Tris-HCl, MgCl2, KCl, and Triton X-100. The total volume of a single PCR reaction tube is generally 20 μl to 200 μl.

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

[0037] Fourthly, a method for the joint detection and differentiation of bloodstream infection pathogens for non-diagnostic purposes is provided, the method comprising the following steps:

[0038] 1) Extract or release nucleic acid from the sample to be tested;

[0039] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition of the present invention as described above or the kit of the present invention as described above;

[0040] 3) Obtain and analyze the results.

[0041] In this invention, the sample used for detection can be blood, plasma, etc., but is not limited to these. It can also be bronchoalveolar lavage fluid, sputum, punctured tissue, pus, wound secretions, etc.

[0042] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0043] DNA pre-denaturation: 95℃ for 1–3 min, 1 cycle; denaturation: 95℃ for 5–20 seconds; annealing: 55℃–60℃ for 10–60 seconds, 30–50 cycles; fluorescence collection.

[0044] In one specific embodiment, use is provided for preparing a composition for the joint detection and differentiation of bloodstream infection pathogens, the joint detection comprising the following steps:

[0045] 1) Extract nucleic acid from the sample to be tested;

[0046] 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition or kit of the present invention as described above;

[0047] 3) Obtain and analyze the results.

[0048] Furthermore, the reaction conditions for the real-time PCR are as follows:

[0049] DNA pre-denaturation: 95℃ for 1–3 min, 1 cycle; denaturation: 95℃ for 5–20 seconds; annealing: 55℃–60℃ for 10–60 seconds, 30–50 cycles; fluorescence collection.

[0050] In this article, the term "non-diagnostic purpose" refers to something not intended to obtain information about whether an individual is infected with the aforementioned pathogens and has a bloodstream infection. For example, the method may be used to detect the presence of the aforementioned pathogens in a test culture (e.g., blood). Attached Figure Description

[0051] Figure 1 The images show the detection results of the compositions of the present invention (specifically, Streptococcus pharyngitis, Streptococcus suis, and Enterococcus faecalis).

[0052] Figure 2 The graph shows the sensitivity results of the composition of the present invention (Streptococcus pharyngitis);

[0053] Figure 3 The graph shows the sensitivity results of the composition of the present invention (slow-onset streptococci);

[0054] Figure 4 This is a graph showing the sensitivity results of the composition of the present invention (Enterococcus faecium);

[0055] Figure 5 This is a graph showing the specificity of the composition of the present invention;

[0056] Figure 6 The image shows the detection results of single-target primers and comparative primers for the composition of the present invention (Streptococcus pharyngitis);

[0057] Figure 7 The image shows the detection results of single-target primers and comparative primers for the composition of the present invention (Streptococcus pyogenes).

[0058] Figure 8 The image shows the detection results (Enterococcus faecium) of the single-target primers and comparative primers of the composition of the present invention.

[0059] Figure 9 The amplification effects of the selected Streptococcus pharyngitis silB-1 and Streptococcus chronicis LTA-1 / LTA-2 combined gene were detected.

[0060] Figure 10 The amplification effects of the selected Streptococcus suis LTA-1 and Streptococcus pharyngitis silB-1 / silB-2 combined gene were detected.

[0061] Figure 11 The amplification effect of the selected Streptococcus pharyngitis silB-1 and Enterococcus faecalis hyl-1 / hyl-2 combined genes was detected. Detailed Implementation

[0062] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0063] Example 1: Primers and probes used in this invention

[0064] The primers and probes used in this invention are shown in Table 1 below.

[0065] Table 1

[0066]

[0067] The fluorescent reporter group for the Streptococcus pharyngitis probe is FAM; the fluorescent reporter group for the Streptococcus suis probe is HEX; and the fluorescent reporter group for the Enterococcus faecium probe is ROX.

[0068] Example 2: Method for detecting Streptococcus pharyngitis, Streptococcus pyogenes, and Enterococcus faecalis.

[0069] Fluorescent PCR amplification reaction solution: contains PCR buffer, Taq enzyme, Mg 2+ The reaction system includes dNTPs, primers, probes, etc. The specific reaction system is shown in Table 2.

[0070] Table 2

[0071] PCR buffer 23.5μL Taq enzyme 2.5 μL (5 U / μL) <![CDATA[Mg 2+ ]]> 0.3 μL (1 M) dNTP 1μL Primer probe 0.25μL Sample DNA 20μL <![CDATA[ddH2O]]> Add to 50μL

[0072] The amplification reaction procedure is shown in Table 3.

[0073] Table 3

[0074]

[0075] Results Analysis and Judgment:

[0076] 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 images, adjust the Start, End, and Threshold values ​​of the Baseline (users can adjust these values ​​according to their actual situation; the Start value can be set between 3 and 15, and the End value between 5 and 20; adjust the amplification curve of the negative control to make it flat or below the threshold line), click Analyze to analyze, and ensure that all parameters meet the requirements in the "Quality Control" section below. Then, record the qualitative results in the Plate window.

[0077] Quality control

[0078] Negative control: No Ct values ​​were displayed in the curves of the FAM, ROX, and HEX channels;

[0079] Positive control: Ct≤36 for the curves of the three channels FAM, ROX, and HEX;

[0080] All of the above requirements must be met simultaneously in the same experiment; otherwise, the experiment is invalid and must be repeated.

[0081] Positive judgment value

[0082] Based on the study of reference values, the Ct reference value for the target gene detected by this kit was determined to be 38.

[0083] Based on the above test results, the judgment results are shown in Table 4 below:

[0084] Table 4

[0085]

[0086]

[0087] Example 3: Detection results of test samples of the composition of the present invention

[0088] The primers and probes shown in Example 1 were used to perform PCR detection of Streptococcus pharyngitis, Streptococcus suis, and Enterococcus faecalis on a real-time PCR instrument according to the method in Example 2. The detection results are as follows: Figure 1 As shown in the figure, the composition of the present invention can effectively detect and distinguish Streptococcus pharyngitis, Streptococcus pyogenes, and Enterococcus faecalis.

[0089] Example 4: Sensitivity of the composition of the present invention

[0090] Using the composition from Example 1 of this invention, LOD (sensitivity) detection was performed on each target to simulate clinical samples, followed by multiplex PCR detection on a quantitative real-time PCR instrument. The results are as follows: Figures 2-4 As shown, this indicates that each channel can still accurately detect samples as low as 500 copies / mL, demonstrating that the sensitivity of the composition of the present invention is 500 copies / mL.

[0091] Example 5: Specificity of the composition of the present invention

[0092] To test the blank specificity of the composition in Example 1 of this invention, a negative control was used as a sample, and the detection was performed according to the above-described operating steps. The results are as follows: Figure 5 The results showed that there was no nonspecific amplification in any of the target channels, indicating good blank specificity of the kit.

[0093] Comparative Example 1: Other primers and probes designed in this invention that do not perform well.

[0094] Due to the principle of complementary base pairing, primers and / or probes can form dimers, but this probability is low and can be eliminated at the initial design stage. However, when detecting multiple pathogens together, there are numerous primers and probes, and dimers can easily form between primers, probes, or between different primers and probes. To ensure the conservation of the design (conservatism is crucial for detection accuracy) while also considering the mutual interference between different primers and probes, careful primer and probe design is required.

[0095] Therefore, the inventors also compared the amplification effects for each target separately, and the results are as follows: Figures 6-8 As shown, different detection systems 1-3 (sequences not shown) were also constructed, which were also used to detect the aforementioned pathogens. Specific detection results are as follows: Figures 9-11 As shown.

[0096] As can be seen from the figure, the differences in amplification effects between the remaining primers and probes are relatively small in single-target detection. However, when placed in a combined detection system, the differences in amplification effects are significantly greater than in single-target detection. This may be due to the mutual influence of primers and probes within the combined detection system; specifically, for example... Figure 8 As shown, the primer and probe pairs designed for Enterococcus faecalis, hyl-1 and hyl-2, showed similar detection effects. However, as... Figure 11 Enterococcus faecalis hyl-1 and hyl-2 were mixed with Streptococcus pharyngitis silB-1 for amplification and detection. The results showed that the combination of hyl-1 and silB-1 was significantly superior to that of hyl-2 and silB-1. Therefore, this demonstrates the superiority of the combined detection system of the present invention. Similarly, the combination of the other two targets also showed similar detection results.

Claims

1. A composition for the joint detection and differentiation of bloodstream infection pathogens, comprising: For example, the upstream primer, downstream primer and probe for detecting Streptococcus pharyngitis shown in SEQ ID NO:1~3; For example, the upstream primer, downstream primer and probe for detecting Streptococcus pyogenes shown in SEQ ID NO:4~6; as well as The upstream primer, downstream primer, and probe for detecting Enterococcus faecalis are shown in SEQ ID NO:7~9.

2. The composition according to claim 1, characterized in that, The composition also includes upstream primers, downstream primers, and probes for detecting the internal standard.

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

4. The composition according to claim 3, characterized in that, The fluorescent reporter group for the Streptococcus pharyngitis probe is FAM; the fluorescent reporter group for the Streptococcus suis probe is HEX; and the fluorescent reporter group for the Enterococcus faecium probe is ROX.

5. The composition according to any one of claims 1 to 4, characterized in that, The components of the composition exist in a mixed form.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a kit for the joint detection and differentiation of bloodstream infection pathogens, wherein, The pathogens are Streptococcus pharyngitis, Streptococcus pyogenes, and Enterococcus faecalis.

7. A kit for the combined detection and differentiation of bloodstream infection pathogens, said kit comprising the composition as described in any one of claims 1 to 5.

8. The reagent kit according to claim 7, characterized in that, The kit also includes negative and positive controls.

9. The reagent kit according to claim 7 or 8, characterized in that, The kit also includes: nucleic acid extraction reagents, DNA polymerase, dNTPs, dUTPs, UDG enzyme, PCR buffer, and Mg. 2+ At least one of them.

10. Use of a reagent for preparing a combined detection and differentiation of bloodstream infection pathogens using the composition of any one of claims 1 to 5 or the kit of any one of claims 7 to 9, the combined detection comprising the following steps: 1) Extract nucleic acid from the sample to be tested; 2) Perform quantitative real-time PCR on the nucleic acid obtained in step 1) using the composition as described in any one of claims 1 to 5 or the kit as described in any one of claims 7 to 9; 3) Obtain and analyze the results.

Images