CN117802254A

CN117802254A - Composition, kit and application for detecting septicemia related pathogens

Info

Publication number: CN117802254A
Application number: CN202311776830.9A
Authority: CN
Inventors: 符小玉; 张登峰; 钟秋梅; 丁峰; 吴康; 刘佳; 戴立忠
Original assignee: Sansure Biotech Inc
Current assignee: Sansure Biotech Inc
Priority date: 2023-12-21
Filing date: 2023-12-21
Publication date: 2024-04-02

Abstract

The invention belongs to the field of molecular biology detection, and particularly relates to detection of related pathogens of septicemia, and more particularly relates to detection of streptococcus angina, stenotrophomonas maltophilia, staphylococcus aureus and enterococcus faecalis. The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that detection and differentiation of streptococcus strainophyllus, stenotrophomonas maltophilia, staphylococcus aureus and enterococcus faecalis are realized in a single-tube reaction system. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection.

Description

Composition, kit and application for detecting septicemia related pathogens

Technical Field

The invention belongs to the field of molecular biological detection, and particularly relates to detection of pathogen related to septicemia, and more particularly relates to streptococcus angina, stenotrophomonas maltophilia, staphylococcus aureus and enterococcus faecalis.

Background

The stenotrophomonas maltophilia (Stenotrophomonas maltophilia) belongs to the xanthomonas family of xanthomonas order and is a gram-negative pathogenic bacterium widely existing in the nature and hospital environment. With the wide application of broad-spectrum antibacterial drugs and immunosuppressants, and the increasing number of invasive procedures, the separation rate of the bacteria in the nonfermentation genus is on the rise. Because of weaker pathogenicity, the infection often occurs in patients with low immunity and critical illness, usually in hospitals, and the community rarely has infection. In addition, the bacteria have inherent drug resistance to various antibacterial drugs, and challenges in clinical anti-infection treatment and infection prevention and control. The most common type of infection is lower respiratory tract infection, which can also cause infections in areas such as the blood stream, urinary system, abdominal cavity, skin and soft tissues. In addition, stenotrophomonas maltophilia often forms mixed infections with other conditional pathogenic bacteria. Infections caused by stenotrophomonas maltophilia can occur in children and adults, particularly in immunocompromised patient populations. Risk factors for infection include: malignant tumor, indwelling central venous catheter, chronic respiratory disease, hypoimmunity, hypoalbuminemia, long-term broad-spectrum antibacterial treatment and long-term hospitalization (especially ICU), etc. Direct contact is the primary mode of transmission, with hands of medical personnel being the most common transmission medium, and susceptible people can be infected by human-to-human and human-to-object surface contact.

The streptococcus angina (Streptococcus anginosus) is one of normal flora planted in the oral cavity, the throat, the digestive tract and the like, and is one of the streptococcus miller. Streptococcus angina is a gram-positive, catalase-negative facultative anaerobic coccus that forms microcolonies on agar medium, belonging to the group of streptococcus grass green. The most common diseases caused by streptococcus angina are respiratory tract infection, suppurative infection and blood flow infection, which account for 34.8%, 33.5% and 8.5% respectively. Previous studies have demonstrated that streptococcus angina produces a variety of toxins in vitro, which often cause invasive suppurative infections, and once invading blood, can circulate throughout the body with blood, causing systemic severe infectious diseases. The blood flow infection of the streptococcus angina group is mostly related to invasive operation, and most patients are combined with basic diseases such as digestive system, cardiovascular system, respiratory system diseases and tumors due to clinical characteristics of the blood flow infection of the streptococcus angina. A plurality of researches show that the most common basic disease in blood flow infection of the streptococcus angina is tumor, and the occupied lesion caused by the tumor is presumed to destroy the normal structure of the human body to cause local infection, so that the streptococcus angina is infected. The streptococcus angina infection features are not obvious, the identification difficulty is high, in clinical work, if the streptococcus angina infection is suspected to be related bacterial infection, the specimen should be examined in time, antibiotics are reasonably applied according to the drug sensitivity result in time, and drug resistance monitoring is carried out so as to improve prognosis. Regarding the identification of streptococcus angina, most countries employ automatic identification instruments and API 20Strep kits, etc. Only a few big hospitals in China can adopt the method, namely 100% of the method can not be used for making correct identification, but the method is still the main identification method of a clinical laboratory.

Staphylococcus aureus (Staphyloccocus aureus Rosenbach) is an important pathogen in humans and is a member of the genus Staphylococcus (Staphylococcus) and can cause a variety of serious infections. There is a generic term "mesophilic bacteria". The typical staphylococcus aureus is spherical and arranged in a cluster under a microscope. Staphylococcus aureus is free of spores, flagella, most of which are capsular and gram-positive. Staphylococcus aureus is ubiquitous in nature and found in air, water, dust and human and animal excretions. Thus, there is a great chance that the food product will be contaminated with it. In recent years, the U.S. center for disease control reports that infection by staphylococcus aureus is the second most second only to escherichia coli. The virulence of staphylococcus aureus is largely dependent on the toxins and invasive enzymes it produces: haemolytic toxin: exotoxins, which are classified into alpha, beta, gamma and delta, can damage platelets, destroy lysosomes and cause ischemia and necrosis of the body; leukocidal hormone: human-destructible white blood cells and macrophages; plasma clotting enzymes: when staphylococcus aureus invades the human body, the enzyme causes fibrin in blood or plasma to be deposited on the surface of cells or to coagulate, thereby inhibiting phagocytosis of phagocytes. Infection by staphylococci is readily localized in relation to this enzyme; deoxyribonuclease: the deoxyribonuclease produced by staphylococcus aureus can resist high temperature and can be used for identifying staphylococcus aureus as a basis; enterotoxin: staphylococcus aureus produces several protein enterotoxins that cause acute gastroenteritis, and are classified into eight serotypes A, B, C, C2, C3, D, E, and F. Enterotoxins can withstand boiling at 100 ℃ for 30 minutes without being destroyed. The symptoms of food poisoning caused by it are vomiting and diarrhea. In addition, staphylococcus aureus also produces lysol, gelatinase, protease, lipase, peptidase, and the like. Staphylococcus aureus is the most common pathogenic bacteria in human suppurative infection, and can cause local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis and the like, and even systemic infection such as septicemia, sepsis and the like.

Enterococcus faecalis (Enterococcus faecalis) is a gram-positive facultative anaerobe of the genus enterococcus. The strain is used as a normal dominant flora in human intestinal tracts, has an important function of maintaining a normal intestinal flora structure, and has stronger tolerance and colonization capacity on intestinal mucosa. Meanwhile, enterococcus faecalis is also a common conditional pathogen, and infection caused by pathogenic enterococcus faecalis includes urinary tract infection, suppurative abdominal infection, septicemia, endocarditis, diarrhea, fever and the like. In addition, enterococcus faecalis has strong environmental adaptability and resistance, and can resist various antibiotics such as tetracycline, kanamycin, gentamicin and the like. Enterococcus faecalis can cause iatrogenic infection, most commonly urinary tract infection caused by urinary tract instrument operation, urethral catheterization and the like, and secondary wounds and surgical postoperative infection of abdominal cavity, pelvic cavity and other parts. Because enterococcus faecalis cell wall thickness, compared with other pathogenic bacteria, the special biological characteristic of the enterococcus faecalis cell wall thickness is more easy to generate drug resistance, and in recent years, the infection caused by enterococcus faecalis is in an ascending trend, and the drug resistance transfer phenomenon occurs, so that the early and timely diagnosis of pathogen infection is of great significance in view of the potential pathogenic risk of enterococcus faecalis.

Sepsis (Wound in) refers to the presence of a sufficient number or toxicity of microorganisms to cause a local or systemic reaction in a host, including persistent pain, red fever, large amounts of exudates, yellow slough >50% of Wound bed, discoloration of granulation tissue, increased tissue vulnerability, susceptibility to bleeding, odor or necrotic tissue. The presence of microorganisms in the wound delays wound healing and increases the risk of amputation and death. Effective prophylactic, diagnostic and therapeutic strategies are critical to reduce mortality and morbidity associated with sepsis.

Therefore, there is a need in the art for a product that can simply and rapidly detect the above pathogens, has high sensitivity and good specificity, provides a relatively sufficient basis for rapid diagnosis and elimination of infection by different pathogens for a clinician, shortens the time for diagnosis of a patient's condition by a clinician, and accelerates the implementation of therapeutic measures to a patient.

Disclosure of Invention

In view of this, in a first aspect, the present invention provides a composition for detecting sepsis-associated pathogens comprising:

an upstream primer, a downstream primer and a probe for detecting streptococcus angina, which are shown in SEQ ID NO. 1-3;

an upstream primer, a downstream primer and a probe for detecting the stenotrophomonas maltophilia, which are shown in SEQ ID NO. 4-6;

an upstream primer, a downstream primer and a probe for detecting staphylococcus aureus, which are shown in SEQ ID NO. 7-9; and

an upstream primer, a downstream primer and a probe for detecting enterococcus faecalis are shown in SEQ ID NO. 10-12.

The joint inspection composition provided by the invention mainly utilizes a multiplex fluorescence PCR analysis method to detect different pathogens by detecting targets on different pathogens, so that streptococcus strainophyllus, stenotrophomonas maltophilia, staphylococcus aureus and enterococcus faecalis are detected and distinguished simultaneously in a single-tube reaction system, and a targeted strategy is provided for subsequent treatment. The composition has higher detection sensitivity reaching 500 copies/mL, good specificity and more accurate detection, provides a basis for a clinician to diagnose and eliminate different pathogen infections more fully and rapidly, shortens the time of the clinician to diagnose the illness state of a patient, and quickens the implementation of treatment measures to the patient.

Further, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

In some specific embodiments, the internal standard is a human internal standard gene. In a specific embodiment, the internal standard is Rnase P.

Further, the fluorophores of the probes of the compositions of the invention are different from each other and do not interfere with each other.

As used herein, "distinct and non-interfering with each other" means that the fluorophores used for each probe in the composition are different and do not affect each other's detection, i.e., can be performed using different channels. For example, ATTO 425, quasar705, FAM, HEX, ROX and CY5 can be used, which groups do not have close absorbance values and can select different channels so as not to interfere with each other.

In some specific embodiments, the fluorescent reporter group of the streptococcus angina probe is FAM; the fluorescent reporter group of the stenotrophomonas maltophilia probe is HEX (or VIC); the fluorescent reporter group of the staphylococcus aureus probe is ROX; the fluorescent reporter group of the enterococcus faecalis probe is CY5.

Further, in some embodiments, the compositions of the present invention may include one or more of the above-described primer and probe pairs simultaneously. In the present invention, "pair" refers to matched upstream and downstream primers and probes that detect a target.

The compositions of the invention can be combined in any combination to detect any combination of 4 targets. Those skilled in the art can combine the primers and probe pairs as necessary to detect which targets are the corresponding targets. These combinations are included in the present invention.

For example, any 3 pairs of the above 4 pairs of primers and probes may be included, any 2 pairs of the above 4 pairs of primers and probes may be included, and any 1 pair of the above 4 pairs of primers and probes may be included.

In some specific embodiments, the compositions of the invention are used in fluorescent PCR.

Further, the 3' end of the probe also has a non-fluorescent quencher.

Further, the 3' -end of the probe also has a quenching group, such as BHQ1 or BHQ2.

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

In a particular embodiment, the ingredients of the composition of the invention are present in separate packages.

In a particular embodiment, the ingredients of the composition of the invention are present in the same package.

Further, the components of the composition of the present invention are present in a mixed form.

In a second aspect, the invention provides the use of a composition of the invention as described above in the manufacture of a kit for detecting a sepsis-associated pathogen, wherein the pathogen is streptococcus angina, stenotrophomonas maltophilia, staphylococcus aureus and/or enterococcus faecalis.

In a third aspect, the invention provides a kit for detecting a sepsis-associated pathogen, the kit comprising a composition of the invention as described above.

Further, the kit also comprises a negative quality control and a positive quality control.

In a specific embodiment, the negative quality control is DEPC H ₂ O, physiological saline. The positive quality control agent is at least one of a segment plasmid or positive strain of streptococcus angina, stenotrophomonas maltophilia, staphylococcus aureus and enterococcus faecalis.

Further, the kit also comprises dNTP, PCR buffer solution and Mg ²⁺ At least one of them.

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

Further, the kit further comprises a nucleic acid releasing reagent, a nucleic acid extracting reagent, dNTPs, dUTP, uracil glycosylase (UDG), a DNA polymerase, a PCR buffer solution and Mg ²⁺ At least one of them.

Further, the concentration of the DNA polymerase is 3U/reaction to 15U/reaction, for example, the DNA polymerase may be Taq enzyme.

In a specific embodiment, the kit of the invention comprises Taq enzyme, UDG enzyme, mg ²⁺ dNTP (U) s, primers, probes and PCR buffer.

Common PCR buffer consists of Tris-HCl and MgCl ₂ Buffer systems such as KCl and Triton X-100. The total volume in a typical single PCR reaction tube is 20. Mu.l to 200. Mu.l.

In a specific embodiment, the kit of the invention is compatible with digital PCR amplification systems, i.e., can be used directly on a digital PCR instrument for amplification.

In a fourth aspect, there is provided a method of detecting sepsis-associated pathogens for non-diagnostic purposes, the method comprising the steps of:

1) Extracting nucleic acid of a sample to be detected;

2) Performing fluorescent quantitative 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;

3) The results were obtained and analyzed.

In the present invention, the sample for detection may be blood, interstitial fluid or the like, but is not limited thereto.

Further, the reaction conditions of the fluorescent quantitative PCR are as follows:

pre-denaturation at 90-95 deg.c for 1-6 min for 1 cycle; denaturation at 90-95 deg.c for 5-20 sec, annealing at 55-60 deg.c for 10-60 sec, 30-50 cycles, and collecting fluorescence.

In a specific embodiment, there is provided the use of a composition for the preparation of a reagent for detecting a sepsis-associated pathogen, the detection comprising the steps of:

1) Extracting nucleic acid of a sample to be detected;

3) The results were obtained and analyzed.

In this context, the term "non-diagnostic purpose" refers to information not intended to obtain whether an individual is infected with the above pathogen and suffering from sepsis. For example, the method may detect the presence of the aforementioned pathogens in culture (e.g., blood, interstitial fluid, etc.) as desired.

Drawings

FIG. 1 shows the results of the detection of the compositions according to the invention (Streptococcus angina, pseudomonas maltophilia, staphylococcus aureus, enterococcus faecalis);

FIG. 2 is a graph showing the sensitivity effect of the composition of the present invention on detecting Streptococcus angina;

FIG. 3 is a graph showing the effect of sensitivity of the composition of the present invention to detect stenotrophomonas maltophilia;

FIG. 4 is a graph showing the effect of sensitivity of a composition of the invention for detecting Staphylococcus aureus;

FIG. 5 is a graph showing the effect of sensitivity of a composition of the invention for detecting enterococcus faecalis;

FIG. 6 is a graph of the results of the specificity of the compositions of the present invention;

FIGS. 7-10 are graphs showing the results of single-check testing of comparative example compositions of the present invention (Streptococcus angina, pseudomonas maltophilia, staphylococcus aureus, enterococcus faecalis, respectively);

FIG. 11 is a graph showing the results of a combination test of Streptococcus strainoxidans silB-1 and Pseudomonas maltophilia sseB-2 in accordance with the comparative example composition of the present invention;

FIG. 12 is a graph showing the results of a combination test of Streptococcus strainotis silB-1 and Staphylococcus aureus splB-1/splB-2 in a comparative example composition of the present invention;

FIG. 13 is a graph showing the results of a combination test of Staphylococcus aureus splB-1 and Pseudomonas maltophilia sseB-1/sseB-2 as comparative compositions according to the present invention;

FIG. 14 is a graph showing the results of a combination test of enterococcus faecalis prgu-1 and Streptococcus strainotis silB-1/silB-2 in a comparative example composition of the present invention;

FIG. 15 is a graph showing the results of a combination test of enterococcus faecalis prgu-1 and Staphylococcus aureus splB-1/splB-2 in a comparative example composition of the present invention.

Detailed Description

The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.

Example 1, primers and probes used in the present invention

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

TABLE 1

Wherein, the fluorescent report group of the streptococcus strainous silB probe is FAM; the fluorescent reporter group of the stenotrophomonas maltophilia SseB probe is HEX; the fluorescent reporter group of the staphylococcus aureus splB probe is ROX; the fluorescent reporter group of the enterococcus faecalis prgu probe is CY5.

Example 2 method for detecting pathogens

Mixing ATCC standard strains corresponding to the four pathogens, diluting the mixed strains with negative whole blood, and detecting the diluted mixed strains serving as samples to be detected. And according to the number of samples to be detected, positive controls and negative controls, mixing corresponding amounts of PCR reaction solutions, fully and uniformly mixing to obtain a PCR mixed solution, centrifuging at 2000rpm for 10s for later use, and configuring a real-time fluorescence PCR reaction system according to the following table 2.

TABLE 2

Sample processing and sample adding:

taking 300 mu L of a sample to be detected, negative control and positive control into a 1.5mL centrifuge tube, and extracting nucleic acid by using a nucleic acid extraction or purification reagent of Sanxiang biotechnology Co., ltd according to the specification operation; and sucking 20 mu L of each of the treated sample, the negative control and the positive control, respectively adding the 20 mu L of each of the treated sample, the negative control and the positive control into a corresponding 0.2mL PCR reaction tube, adding 30 mu L of PCR mixed solution into each tube, and covering a tube cover.

And (3) PCR amplification:

PCR amplification was performed by using a PCR apparatus such as the biological system 7500 according to the temperature and time setting program shown in Table 3.

TABLE 3 Table 3

Analysis of results:

1) The target detection signal is FAM, HEX (or VIC), ROX and CY5;

2) Setting of Baserine: baseline is typically set to 3-15 cycles, which can be specifically adjusted according to the actual situation. The adjustment principle is as follows: the region where the fluorescent signal is more stable before exponential amplification is selected, the starting point (Start) avoids the signal fluctuation in the initial stage of fluorescent collection, and the End point (End) is reduced by 1-2 cycles compared with the sample Ct of which the exponential amplification occurs at the earliest. Setting of Threshold: setting a principle that a threshold line just exceeds the highest point of a normal negative control;

3) Quality control negative control: the curves of the four channels FAM, HEX, ROX, CY are all without Ct values;

positive control: FAM, HEX, ROX, CY5 curve Ct of three channels is less than or equal to 38;

the requirements are met in the same experiment, otherwise, the experiment is invalid and needs to be carried out again.

Positive judgment value

The Ct reference value of the target gene detected by the kit is determined to be 38 through the research of the reference value.

4) Interpretation of results

TABLE 4 Table 4

Example 3 detection results of test samples of the inventive composition

The primers and probes shown in example 1 were used to verify samples of Streptococcus angina, pseudomonas maltophilia, staphylococcus aureus and enterococcus faecalis in the manner of example 2, and PCR was performed on a macrostone fluorescent quantitative PCR apparatus, the detection results are shown in FIG. 1, and it can be seen from the graph that the composition of the present invention can well detect and distinguish these 4 pathogens.

Example 4 sensitivity of the composition of the invention

Using the composition of example 1 of the present invention, LOD (sensitivity) detection was performed on each target at concentrations of 1000, 800, 500 copies/ml, respectively, to simulate clinical samples, and 20 multiplex PCR assays were performed on a macrostone fluorescent quantitative PCR instrument. The results are shown in FIGS. 2-5, which show that each channel can still be accurately detected for samples as low as 500 copies/mL, and the detection rate is 100%, which shows that the sensitivity of the composition of the invention is 500 copies/mL.

EXAMPLE 5 specificity of the composition of the invention

In order to test the blank specificity of the composition of example 1 of the present invention, a negative control was used as a sample, and the test was performed according to the procedure described above. As shown in FIG. 6, each target channel has no non-specific amplification, and the blank specificity of the kit is good.

Comparative example 1, remaining poorly performing primers and probes designed according to the invention

Because of the base-pairing rules, dimers are formed between the primer and/or probe, but with little probability, this can be eliminated at the beginning of the design. However, when multiple pathogens are jointly detected, a plurality of primers and probes are arranged, dimers are easy to occur between the primers and the primers, between the probes and the probes or between the primers and the probes, so that the conservation of design (which is crucial to the accuracy of detection) is ensured, and the mutual interference among different primer probes is considered, so that the primer probes need to be carefully designed.

Thus, the inventors have devised that the remaining primers and probes constitute a different detection system (sequences not shown) for detecting the above pathogens as well. Specific detection results are shown in fig. 7-10, and it can be seen from the figures that the difference of detection effects of different primers and probes for detecting 4 targets in a single detection system is small, however, detection is affected in a four-joint detection system, and obvious layering of an amplification curve and remarkable reduction of fluorescence increment exist, as shown in fig. 11-15, which are probably caused by the influence of the primers and probes of the joint detection system, so that the superiority of the composition of the invention is further illustrated.

Claims

1. A composition for detecting a sepsis-associated pathogen comprising:

2. The composition of claim 1, wherein the fluorophores of the probes of the composition are different from each other and do not interfere with each other.

3. The composition of claim 1, further comprising an upstream primer, a downstream primer, and a probe for detecting an internal standard.

4. The composition of claim 3, wherein the fluorescent reporter group of streptococcus angina is FAM; the fluorescent reporter group of the stenotrophomonas maltophilia is HEX or VIC; the fluorescent reporter group of staphylococcus aureus is ROX; the fluorescent reporter group of the enterococcus faecalis probe is CY5.

5. The composition according to any one of claims 1 to 4, wherein the components of the composition are present in a mixed form.

6. Use of a composition according to any one of claims 1 to 5 for the preparation of a kit for detecting a sepsis-associated pathogen, wherein the pathogen is streptococcus angina, stenotrophomonas maltophilia, staphylococcus aureus and/or enterococcus faecalis.

7. A kit for detecting a sepsis-associated pathogen, the kit comprising the composition of any one of claims 1-5.

8. The kit of claim 7, further comprising a negative quality control and a positive quality control.

9. The kit of claim 7 or 8, further comprising: nucleic acid releasing reagent, nucleic acid extracting reagent, DNA polymerase, dNTP, dUTP, UDG enzyme, PCR buffer solution and Mg ²⁺ At least one of them.

10. Use of a composition for the preparation of a reagent for detecting a sepsis-associated pathogen, the detection comprising the steps of:

1) Extracting nucleic acid of a sample to be detected;

2) Performing fluorescent quantitative PCR on the nucleic acid obtained in step 1) using the composition of any one of claims 1 to 5 or the kit of any one of claims 7 to 9;

3) The results were obtained and analyzed.