Digital PCR (polymerase chain reaction) kit for detecting bloodstream infection pathogens

Abstract

The invention provides a digital PCR kit for detecting bloodstream infection pathogens, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli and stenotrophomonas maltophilia are simultaneously detected in a tube of the kit, and a buffer system of the kit comprises Tris-HCl, glycerol, dNTPs, KCl, MgCl2, BSA, SSB, Taq DNA polymerase, UNG enzyme, primers and probes. The volume concentration of glycerol in the buffer system is 4%-8%, the mass volume concentration of BSA in the buffer system is 0.2%-0.8%, and the concentration of SSB in the buffer system is 2-4 ng / L. In the buffer system, the stability of the liquid drops is improved, the amplification efficiency is improved, low-concentration and high-concentration targets can be stably detected, when a large volume of nucleic acid template is added, the liquid drops are stable, the amplification efficiency is not influenced, and the clinical use effect is met.

Description

Technical Field

[0001] The present invention relates to the field of digital PCR detection, and in particular to a digital PCR kit for detecting bloodstream infection pathogens. Background Art

[0002] Bloodstream infection (BSI) is a systemic inflammatory response syndrome (SIRS) caused by the invasion of various pathogenic microorganisms and their toxins into the blood circulation. Pathogenic microorganisms exist transiently, intermittently or persistently in the circulating blood. As a serious systemic infectious disease, it is prone to induce sepsis and multiple organ dysfunction syndrome, with a high mortality rate. It has become one of the major public health burdens worldwide. The prognosis of patients with bloodstream infection is closely related to the timeliness and accuracy of diagnosis and the rational use of initial antimicrobial drugs.

[0003] At present, the detection methods of pathogens in clinical practice are roughly divided into blood culture, sequencing technology, antigen detection methods, and molecular detection methods. Blood culture (BC) is the gold standard for pathogen detection and is cheap. However, blood culture also has obvious disadvantages such as complex operation, long culture cycle, high requirements for experimental conditions, difficulty in culturing some pathogens, and great influence of antibiotic treatment. Sequencing technology has the characteristics of comprehensive and broad spectrum coverage, but its cost is high, requiring higher-level supporting instruments, reagents, and professional operators, and the experimental operation is complicated and the detection time is relatively long, so it is difficult to popularize it widely. The advantages of antigen detection technology are fast, simple operation, and low price, but it has disadvantages such as subjective result reading, great influence of antibiotic treatment, and latex agglutination test relying on antigen expression.

[0004] Molecular diagnostic technology is based on primer amplification of target pathogens. Its detection does not rely on live bacteria, so it has high sensitivity and specificity. Although qPCR also has the above detection advantages, its interference ability is weak, and if it is to be quantified, it must rely on the detection of standard products. However, ddPCR, while having the above molecular diagnostic advantages, can well make up for the shortcomings of qPCR. ddPCR can achieve absolute quantification in the true sense, has strong anti-interference ability, can tolerate the influence of PCR inhibitors, and is suitable for the detection of a variety of complex samples. The price is much cheaper than sequencing. The key is that it can give test results within 3-4 hours, so it can well meet the current clinical needs. At present, combined with the detection speed, detection cost, detection operation steps and large-scale popularization, it can be seen that ddPCR is the best detection method for pathogen detection. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a digital PCR kit for detecting bloodstream infection pathogens, wherein the kit simultaneously detects Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Stenotrophomonas maltophilia in one tube, and the buffer system of the kit includes Tris-HCl, glycerol, dNTPs, KCl, MgCl2, Taq DNA polymerase, UNG enzyme, primers and probes, the volume concentration of glycerol in the buffer system is 4%-8%, the mass volume concentration of BSA in the buffer system is 0.2%-0.8%, and the SSB concentration in the buffer system is 2-4ng / µL.

[0006] In one embodiment, the mass volume concentration of BSA in the buffer system is 0.2%-0.4%.

[0007] In one embodiment, the SSB concentration in the buffer system is 2-3 ng / µL.

[0008] In one embodiment, the buffer system of the kit includes Tris-HCl, glycerol, dNTPs, KCl, MgCl2, Taq DNA polymerase, UNG enzyme and primers and probes, wherein the volume concentration of glycerol in the buffer system is 4%, the mass volume concentration of BSA is 0.4%, the concentration of SSB is 3ng / µL, the concentration of magnesium ions is 6mM, and the concentration of Taq enzyme is 3U.

[0009] In one embodiment, the kit contains the following primers and probes: .

[0010] In one embodiment, the final concentrations of the primers and probes for Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia, and Klebsiella pneumoniae in the buffer system of the kit are 500 nM and 250 nM, respectively.

[0011] In the present invention, three sets of primers and probes are designed for Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia and Klebsiella pneumoniae, respectively, and the preferred primers and probes for each pathogen are screened on this basis, and then the tube test is carried out to obtain the best primer and probe combination after the tube. After the combination, in the high-sensitivity bloodstream infection pathogen detection, since a large volume of plasma samples is required for nucleic acid extraction, and the amount of nucleic acid template added in the system needs to be as large as possible, the stability of the droplets is a key issue. In the extraction process of large-volume plasma samples (1-5ml, more plasma in some cases), and when a large volume of nucleic acid template is added, the formation and stability of the droplets may be interfered by the residual plasma components in the extracted nucleic acid, such as high-viscosity substances, proteins or other particulate matter in the plasma, which can easily cause the droplets to break or merge, thereby affecting the accuracy and repeatability of the test results.

[0012] When a large volume of plasma is used to extract nucleic acid for detection, there are no or fewer droplets, and the combined tube copy number of each target is lower than the expected target copy number. When a large volume of nucleic acid is used for detection, the droplet stability decreases and the amplification efficiency is reduced. In order to improve the droplet stability and the amplification efficiency after combining the tubes, the present invention optimizes the buffer system. In the buffer system of the present invention, the volume concentration of glycerol is 4%-8%, and the mass volume concentration of BSA is 0.2%-0.8%. When the template concentration is high, the quantitative results are in line with expectations, indicating that the amplification efficiency is improved, but when the template concentration is low, the quantification is still low. When the SSB concentration is 1 ng / μl, the quantitative results of low-copy templates are low, and the quantitative results of high-copy templates are basically in line with expectations. When the SSB concentration is increased to 2 ng / μl-4 ng / μl, the quantitative results of low-concentration templates and high-concentration templates are significantly increased, and the expected copy number is also increased by more than 50%, indicating that SSB stabilizes the single-stranded template and disperses it into different droplets, thereby improving the detection efficiency. When SSB is increased to 5 ng / μl, the competitive binding between the primer and the template is affected due to the high amount of SSB, and the quantitative copy number is reduced at this time. Therefore, the more suitable SSB concentration is 2 ng / μl-4 ng / μl, and 2 ng / μl-3 ng / μl is preferred. In the buffer system of the present invention, through the coordination between glycerol, BSA and SSB, the droplet stability of the buffer system is improved, the amplification efficiency is improved, the nucleic acid extracted from plasma can be stably detected, and when the nucleic acid template is added in a large volume, the droplet is stable, the amplification efficiency is not affected, and the low-concentration and high-concentration targets are stably detected, meeting the clinical use effect. DETAILED DESCRIPTION

[0013] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the embodiments described are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0014] Example 1 Primer probe screening Three pairs of primer probes were designed for the specific genes of the bacteria to be detected (Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Stenotrophomonas maltophilia), and the primer probes were screened using the basic system. Table 1 below shows the candidate primer probe sequences.

[0015] Table 1. Primer and probe information

[0016] 1. Preparation of basic PCR reaction system Prepare the basic PCR reaction system according to the following table

[0017] The final concentration of primers was 500 nM, and the final concentration of probes was 250 nM. The templates used nucleic acids extracted from Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Stenotrophomonas maltophilia (1 ml of bacterial culture was extracted using the Xinyi Free DNA Nucleic Acid Extraction Kit), the nucleic acids were quantified and diluted, and then about 3000 copies / system were added to the system. In addition, two mixed nucleic acids were used as cross-reaction templates, including 1) Cross-reaction template 1 (including nucleic acids from 4 other pathogens, each with a concentration of 2000 copies / µL) to evaluate the cross-reaction specificity between primers and probes; 2) Cross-reaction template 2 (mixed with nucleic acids from multiple other pathogens, including HIV, EBV, VZV, BKV, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Haemophilus influenzae, and Candida albicans, each with a concentration of 2000 copies / µL) to evaluate the specificity of primers and probes to other pathogens.

[0018] 2. Digital PCR Workflow 2.1. Preparation of microdroplets: Using a droplet generation chip (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) and a sample preparation instrument (Xinyi Manufacturing Technology (Beijing) Co., Ltd.), 30 µL of PCR reaction system was added to the sample well of the droplet generation chip, and 180 µL of droplet generation oil was added to the oil well. The chip and 8-strip tubes were placed in the preparation instrument, covered with a rubber pad, and microdroplet preparation was performed.

[0019] 2.2. PCR amplification: Place the 8-tube strip containing microdroplets on a PCR instrument for amplification. The amplification program is set as shown in the following table:

[0020] 2.3. Droplet detection: After the PCR is completed, place the 8-tube strip and droplet detection chip (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) into the fixture, add 430 µL and 500 µL detection oil to the oil wells respectively, cover with a rubber pad, and place the chip into the chip analyzer (Xinyi Manufacturing Technology (Beijing) Co., Ltd.) for droplet detection.

[0021] 2.4. Data analysis: Each of the tens of thousands of microdroplets prepared by the droplet generation chip and the sample preparation instrument is an independent PCR reactor. Most of the microdroplets do not contain the target gene to be tested or contain at least one target gene to be tested. After PCR amplification, the chip analyzer detects the fluorescence signal of each channel of each microdroplet, records the peak height of the microdroplet signal, and the droplets containing the target gene to be tested will be detected with the corresponding fluorescence signal. The fluorescence intensity in the microdroplet is digitized by the fluorescence classification threshold. The microdroplets with strong fluorescence are judged as "1" (positive), and the microdroplets with weak fluorescence are judged as "0" (negative). The number of "1" and "0" is counted, and the total number of copies of each fluorescently labeled target gene in the input template can be calculated through the Poisson distribution model correction.

[0022] 3. Filter results The working condition of the primers and probes was examined based on whether amplification occurred, the ratio of the positive signal to the background signal (signal-to-noise ratio) and the copy number. The screening results for each target are shown in Table 2. For each target, at least 1-2 groups of primer and probe combinations that can be specifically amplified and whose copy numbers meet expectations were screened.

[0023] Table 2 Screening results for each target

[0024] Example 2: Testing of different targets in parallel The primer-probe combination for single-plex amplification screened above was tested in parallel according to the following table, wherein the final concentration of the primer was 500 nM, and the final concentration of the probe was 250 nM.

[0025]

[0026] The specific PCR reaction system preparation and digital PCR workflow are the same as in Example 1. The nucleic acid mixture extracted from Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Stenotrophomonas maltophilia is used as a template to determine the parallel detection situation. In addition, the cross-reaction template 2 in Example 1 is used to determine the specificity after paralleling. The screening results are shown in Table 3. The parallel test results show that Klebsiella pneumoniae in combination 1 and combination 3 were not amplified normally, there was non-specific amplification in combination 2, and normal amplification in combination 4, and the amplification signal was basically consistent with the expected result. Therefore, combination 4 is used as the subsequent parallel combination, and the primer probe combination screened after the parallel combination is shown in Table 4.

[0027] Table 3 Results of parallel pipe screening test

[0028] Table 4. Primer-probe combinations screened after combined tube combination

[0029] Example 3 Screening of different detection systems In the above-mentioned embodiments, the nucleic acid used in the primer-probe screening and primer-probe combination screening is the nucleic acid extracted from the bacterial culture, and the amount of template added to the system is 1µl. In the high-sensitivity bloodstream infection pathogen detection, a large volume of plasma samples is required for nucleic acid extraction, and the amount of nucleic acid template added to the system needs to be as large as possible, so the stability of the droplets is a key issue. During the extraction process of large-volume plasma samples (1-5ml, more plasma in some cases), and when a large volume of nucleic acid template is added, the formation and stability of the droplets may be interfered by the plasma components remaining in the extracted nucleic acid, such as high-viscosity substances, proteins or other particulate matter in the plasma, which can easily cause the droplets to break or fuse, thereby affecting the accuracy and repeatability of the test results. According to reports, the concentration of infectious pathogens in plasma is generally 10-10000 copies of pathogens / ml plasma. In order to achieve a sensitivity of 10 copies of pathogens / ml, this embodiment uses 1ml simulated plasma sample (5 pathogen nucleic acids are mixed, including low-concentration 20 copies / ml plasma and medium-high concentration 3000 copies / ml plasma, and the bacterial solution is mixed into the plasma matrix) for extraction, and the nucleic acid elution volume is 40μL (a smaller elution volume is used to concentrate the nucleic acid concentration as much as possible), and then a large volume of nucleic acid is added to the system to ensure sensitivity. In this embodiment, 20μL of nucleic acid template is added, and the detection system in Example 1 is used as the basic system to optimize the detection system to optimize and screen out a detection system that ensures droplet stability and high amplification efficiency. In order to increase the strength of the droplet film, it is necessary to wrap a layer of highly viscous liquid on the surface of the droplet so that the droplet is not easy to break and merge when the pressure fluctuates. Increasing the amount of glycerol or using BSA (bovine serum albumin) and other measures can prevent droplet merging and aggregation and ensure the dispersibility of droplets. Among them, glycerol is a highly viscous liquid, and BSA can reduce the nonspecific adsorption of nucleic acids or reagents and improve sensitivity. BSA further enhances droplet stability in synergistic effect with high viscosity liquid.

[0030] In addition, since the present invention detects pathogen free nucleic acids in the blood, the content of pathogen nucleic acids is relatively low, as mentioned above. Therefore, in order to further improve the detection sensitivity, a single-stranded binding protein (SSB) is additionally added to the system. SSB binds to single-stranded DNA to prevent it from back-chaining or forming a secondary structure. For plasma free DNA (cfDNA), the concentration is low and the fragments are relatively short. SSB can prevent these short fragments from renaturing after denaturation, thereby preventing the primer binding efficiency from being reduced.

[0031] Furthermore, in droplet PCR detection, since SSB can stabilize the single-stranded structure of the template, the single-stranded nucleic acid template is distributed in different droplets, further increasing the detection copy number. Compared with the detection situation where the double-stranded template enters the same droplet under normal circumstances, the copy number can theoretically be doubled. SSB can also reduce the occurrence of nonspecific amplification, especially in complex backgrounds (such as other DNA or proteins present in plasma). In summary, SSB can improve the amplification efficiency by stabilizing single-stranded templates and polymerases, especially in the detection of plasma free DNA at low template concentrations. However, excessively high concentrations of SSB may inhibit primer annealing, thereby reducing PCR efficiency. Therefore, the optimal concentration of SSB needs to be optimized for a specific reaction system.

[0032] The volume of microdroplets generated by the digital PCR platform and supporting consumables (sample preparation instrument, chip analyzer, supporting microdroplet generation chip and generation oil, microdroplet detection chip and detection oil) of Xinyi Manufacturing Technology (Beijing) Co., Ltd. is about 0.5nL. The theoretical number of microdroplets generated by a 30μL reaction system is about 60,000. When detecting and analyzing microdroplets, the more droplets detected (but should not exceed the theoretical number of droplets generated), the higher the sensitivity and stability of the detection. Especially in the detection of low-copy targets (less than 50 copies in the system), if the target-containing droplets are lost, the quantitative results will be low. When the target copy number is high (higher than 50 copies), the detection result is corrected by the Poisson distribution formula. The number of detected droplets has little effect on the quantitative result, but it is usually required to detect at least 50% of the generated droplets for the detection result to be stable and reliable, that is, the number of detected droplets reaches more than 30,000, and the detection result meets the standard.

[0033] (1) Optimize the glycerol concentration. The system preparation is shown in Table 5. There are 5 systems in total, and the final glycerol concentrations are 1%, 2%, 4%, 8% and 10% respectively. The test results are shown in Table 6. When the glycerol concentration is 1%, the droplets merge and no droplets are detected; at a concentration of 2% and above, droplets can be detected, and the number of droplets detected increases with the increase of glycerol concentration. When the glycerol concentration reaches 4% and above, the number of droplets is more than 30,000, but when the glycerol concentration is 10%, the number of droplets reaches 70,000, which exceeds the theoretical number of droplets. Therefore, the glycerol concentration of 4%-8% meets the droplet number requirements, but the detection copy number is low at low copies, indicating that the amplification efficiency is affected when the template input amount (20µL) is increased, and further optimization is needed.

[0034] Table 5. Preparation of detection system with different glycerol concentrations

[0035] Table 6. Test results of different glycerol concentrations

[0036] (2) Optimize the BSA concentration. The system preparation is shown in Table 7. There are 5 systems in total. Under the condition of a fixed glycerol concentration of 4%, systems with different BSA concentrations were prepared. The final BSA concentrations were 0.1%, 0.2%, 0.4%, 0.8% and 1%, respectively. The test results are shown in Table 8. The number of droplets in different systems is relatively normal, ranging from 40,000 to 60,000. When the BSA concentration is 0.1%, the quantitative results are low for both low-copy templates and high-copy templates, which may be due to insufficient amplification efficiency. When the BSA concentration is increased to 0.2%-0.8%, the quantitative results are in line with expectations for high-concentration templates, indicating that the amplification efficiency is improved. However, the quantitative results are still low for low-concentration templates, indicating that for the detection of plasma pathogen free DNA, the detection efficiency needs to be further improved for low-concentration templates. When the BSA is increased to 1%, the amplification conditions of the taq enzyme are affected due to the high amount of BSA. At this time, the quantitative copy number decreases instead. Therefore, the more suitable BSA concentration is 0.2%-0.8%, especially 0.2%-0.4%.

[0037] Table 7. Preparation of detection system with different BSA concentrations

[0038] Table 8. Detection results of different BSA concentrations

[0039] (3) Optimize the SSB concentration. The system preparation is shown in Table 9. There are 5 systems in total. Under the conditions of fixed glycerol concentration of 4% and BSA concentration of 0.4%, systems with different SSB concentrations were prepared. The final SSB concentrations were 1 ng / μl, 2 ng / μl, 3 ng / μl, 4 ng / μl and 5 ng / μl, respectively. The test results are shown in Table 10. The number of droplets is relatively normal in different systems, ranging from 40,000 to 60,000. When the SSB concentration is 1 ng / μl, the quantification of low-copy templates is low, and the quantification of high-copy templates is basically in line with expectations, indicating that the SSB concentration is not enough. When the SSB concentration is increased to 2 ng / μl-4 ng / μl, the quantitative results of both low-concentration templates and high-concentration templates are significantly increased, and the expected copy number is also increased by more than 50%, indicating that SSB stabilizes the single-stranded template and disperses it into different droplets, thereby improving the detection efficiency. When SSB is increased to 5 ng / μl, the competitive binding between the primer and the template is affected due to the high amount of SSB, and the quantitative copy number decreases instead. Therefore, the more suitable SSB concentration is 2 ng / μl-4 ng / μl, especially 2 ng / μl-3 ng / μl.

[0040] Table 9. Preparation of detection system with different SSB concentrations

[0041] Table 10. Test results of different SSB concentrations

[0042] (4) Optimize other components in the detection system The concentration of glycerol in the fixed system was 4%, the concentration of BSA was 0.4%, and the concentration of SSB was 3 ng / μl. The concentrations of other components in the multiplex system were optimized. 2+ The concentrations include 4mM, 6mM, and 8mM; the Taq enzyme concentrations include 2U, 3U, and 4U. The system preparation table refers to system 13 in Table 9. According to system 13, the concentrations of Mg2+ and Taq enzyme were changed for preparation. There are 8 combinations in total, see Table 11-1 and Table 11-2. When screening each optimized combination, multiple system combinations are evaluated and screened from aspects such as amplification efficiency, copy number, and total droplet number (droplet stability).

[0043] Table 11-1. Preparation of detection system with different Taq enzyme amounts and MgCl2 concentrations

[0044] Table 11-2. Preparation of detection system with different Taq enzyme amounts and MgCl2 concentrations

[0045] Using system 13 as a benchmark, systems 16-23 were prepared to detect nucleic acid mixtures extracted from simulated plasma of 5 pathogens, respectively. The template nucleic acid was added in an amount of 20µL, and a high-copy template (about 2000 copies / system) was used. The specific PCR reaction system preparation and digital PCR workflow were the same as above, and the screening results are described below.

[0046] (1) Number of droplets Count the total number of droplets and evaluate whether the system has an impact on droplet stability.

[0047]

[0048] From the above results, it can be seen that different systems have little effect on the stability of droplets. The total number of droplets in the eight systems is greater than 30,000. The results are all reliable and will be further analyzed later.

[0049] (2) Amplification efficiency assessment The nucleic acid template in this experimental group is a high-concentration nucleic acid (the template is added in a volume of 20µL), and the copy number quantitative results are counted to evaluate the amplification efficiency of the system. The results are shown in the following table:

[0050] From the above results, we can see that system 19 (glycerol concentration is 4%, BSA concentration is 0.4%, SSB is 3ng / µL, Mg2+ When the concentration was 6mM and the Taq enzyme concentration was 3U), the copy number of each target was optimal. And according to the test results, there was no significant difference in the fluorescence signal difference among the systems. Based on the above results, the final system was determined to be system 19, with a glycerol concentration of 4%, a BSA concentration of 0.4%, a SSB concentration of 3ng / µL, and a Mg 2+ The concentration was 6 mM and the Taq enzyme concentration was 3 U.

[0051] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0052] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A digital PCR kit for detecting bloodstream infection pathogens, characterized in that: The kit can simultaneously detect Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Stenotrophomonas maltophilia in one tube. The buffer system of the kit includes Tris-HCl, glycerol, dNTPs, KCl, MgCl2, Taq DNA polymerase, UNG enzyme, primers and probes. The volume concentration of glycerol in the buffer system is 4%-8%, the mass volume concentration of BSA in the buffer system is 0.2%-0.8%, and the concentration of SSB in the buffer system is 2-4 ng / µL.

2. The digital PCR kit according to claim 1, characterized in that: The mass volume concentration of BSA in the buffer system is 0.2%-0.4%.

3. The digital PCR kit according to claim 2, characterized in that: The SSB concentration in the buffer system is 2-3 ng / µL.

4. The digital PCR kit according to claim 3, characterized in that: The buffer system of the kit includes Tris-HCl, glycerol, dNTPs, KCl, MgCl2, Taq DNA polymerase, UNG enzyme, primers and probes. The volume concentration of glycerol in the buffer system is 4%, the mass volume concentration of BSA is 0.4%, the concentration of SSB is 3ng / µL, the concentration of magnesium ions is 6mM, and the concentration of Taq enzyme is 3U.

5. The digital PCR kit according to claim 1, characterized in that: The kit contains the following primers and probes: 。 6. The digital PCR kit according to claim 5, characterized in that: The final concentrations of the primers and probes of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia and Klebsiella pneumoniae in the buffer system of the kit are 500 nM and 250 nM respectively.

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