A kit and method for detecting blood stream infection bacterial nucleic acid by using multiple probe melting curve method

Abstract

The application discloses a kit and a method for detecting blood stream infection bacterial nucleic acid by using a multiple probe melting curve method, and utilizes a multiple probe melting curve technology to adopt four different fluorescent probes to carry out fluorescent PCR combined with multiple melting curves, and develops an eight-target bacterial detection kit for common eight kinds of bacteria in blood stream infection. The application has the sensitivity and the specificity of the fluorescent quantitative PCR, and has a larger detection flux than the fluorescent quantitative PCR method, is more convenient to operate, and is lower in cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a kit and method for detecting blood infection bacterial nucleic acid by using multiplex probe melting curve method. BACKGROUND

[0002] Sepsis refers to various pathogenic bacteria entering blood circulation and proliferating and spreading in the blood circulation, producing toxins and metabolites, leading to systemic infection, and causing serious adverse consequences. If not treated in time, it can endanger life, and is a common and high-mortality disease in childhood infectious diseases. The treatment of sepsis in children mainly depends on the use of corresponding antibiotics. The antibiotics used for different pathogenic bacteria are quite different, and the symptoms and prognosis of sepsis caused by different pathogenic bacteria are also quite different. Therefore, early and accurate diagnosis of sepsis in children is particularly important.

[0003] Combined with literature retrieval and historical data of our hospital, we found that the main bacteria of blood stream infection in children include Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae and Enterococcus.

[0004] In clinical practice, blood culture is the gold standard for diagnosing sepsis in children, but blood culture is easy to be contaminated, resulting in high false positive rate, many bacteria are difficult to culture, resulting in high false negative rate, and the culture time is long. The shortcomings such as time-consuming still exist. Fluorescent quantitative PCR detection is the most widely used technical method in clinical practice at present. The main advantages of this method are high sensitivity and high specificity. However, the detection throughput of this method is limited by the characteristics of fluorescent substances. The commonly used detection fluorescent quantitative PCR detection kit in clinical practice can only detect 1-2 kinds of bacteria, which cannot meet the demand of clinical screening of multiple bacteria. SUMMARY

[0005] The first object of the present application is to provide a kit for detecting blood infection bacterial nucleic acid by using multiplex probe melting curve method, which solves the above-mentioned problems in the prior art. The second object of the present application is to provide a method for detecting blood infection bacterial nucleic acid by using the above-mentioned kit.

[0006] As one aspect of the present application, the present application provides a kit for detecting blood infection bacterial nucleic acid by using multiplex probe melting curve method, which comprises a fluorescent PCR reaction solution and a DNA polymerase solution. The fluorescent PCR reaction solution contains a PCR buffer, MgCl2, dNTPs, an upstream primer, a downstream primer, and 8 kinds of bacterial target corresponding fluorescent probes. The 8 kinds of bacterial targets are gram-negative bacteria, gram-positive bacteria, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae, and Enterococcus.

[0007] The upstream primer sequences are shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, and SEQ ID NO: 20.

[0008] The downstream primer sequences are shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21.

[0009] The fluorescent probe sequences corresponding to the eight bacterial targets are shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22.

[0010] As a preferred embodiment of the kit for detecting bloodstream bacterial nucleic acid using the multiple probe melting curve method described in this invention, it further includes a positive control and a negative control. The positive control is a mixture of the amplified sequences of the eight bacterial targets dissolved in TE buffer, and the negative control is sterile water.

[0011] As a preferred embodiment of the kit for detecting bloodstream bacterial nucleic acid using the multiple probe melting curve method described in this invention: the DNA polymerase solution is Taq DNA polymerase; the PCR buffer is TE buffer with pH = 8.2.

[0012] As another aspect of the present invention, the present invention also provides a PCR method for detecting nucleic acids of bloodstream-infecting bacteria using the kit described herein, comprising the following steps:

[0013] Step S01, Nucleic acid extraction: Extract nucleic acid from the sample to be tested using magnetic bead extraction or column extraction method;

[0014] Step S02, reagent preparation: Prepare fluorescent PCR reaction solution and DNA polymerase solution, mix them to obtain PCR qualitative reaction solution;

[0015] Step S03, Amplification and Detection: Take one of the nucleic acid, positive control, and negative control obtained in step S01 and add them to the PCR qualitative reaction liquid obtained in step S02 to obtain the PCR reaction system. Detect the system using a fluorescence quantitative PCR instrument with 4 or more colors.

[0016] Step S04: Perform fluorescence quantitative analysis.

[0017] As a preferred embodiment of the method described in this invention: in step S02, 19 μL of fluorescent PCR reaction solution and 1 μL of DNA polymerase solution are taken to prepare 20 μL of PCR qualitative reaction solution.

[0018] As a preferred embodiment of the method described in this invention: In step S03, 5 μL of nucleic acid, positive control, or one of the positive control samples is added to the 20 μL PCR qualitative reaction liquid obtained in step S02 to obtain a 25 μL PCR reaction system.

[0019] As a preferred embodiment of the method described in this invention: the concentration of DNA polymerase in the PCR qualitative reaction liquid is 5U; the concentration of MgCl2 is 4mM; and the concentration of dNTP is 10mM.

[0020] As a preferred embodiment of the method described in this invention, the PCR reaction program is as follows: 95℃ for 5 min, 1 cycle; 95℃ for 5 s, 58℃ for 30 s, 30 cycles; 95℃ for 5 s, 60℃ for 30 s, 20 cycles; 95℃ for 10 s, 1 cycle; 60℃~85℃ at a rate of 0.03℃ / s, 1 cycle; 37℃ for 15 s.

[0021] As a preferred embodiment of the method described in this invention: when the CT values ​​of the FAM probe are all greater than 25, the CT values ​​of the VIC probe are all greater than 30, the CT values ​​of the ROX probe are all greater than 30, and the CT values ​​of CY5 are all greater than 30, then it is a negative specimen;

[0022] When the Ct value of the FAM fluorescence channel is ≤25, it indicates that the bacteria are either Gram-negative or Gram-positive. Further analysis of the melting curve shows that a melting peak at 69.0±1℃ indicates Gram-positive bacteria, and a melting peak at 76.0±1℃ indicates Gram-negative bacteria.

[0023] When the Ct value of the VIC fluorescence channel is ≤30, it indicates Staphylococcus aureus or Klebsiella pneumoniae. Further analysis of the melting curve shows that a melting peak at 72.0±1℃ indicates Staphylococcus aureus positivity, and a melting peak at 63±1℃ indicates Klebsiella pneumoniae positivity.

[0024] When the Ct value of the ROX fluorescence channel is ≤35, it indicates that the bacteria are positive for Streptococcus pneumoniae, Streptococcus agalactiae, and Enterococcus. Further analysis of the melting curve shows that a melting peak at 66.0±1℃ indicates a positive result for Streptococcus agalactiae, a melting peak at 69.0±1℃ indicates a positive result for Streptococcus pneumoniae, and a melting peak at 77.0±1℃ indicates a positive result for Enterococcus.

[0025] When the Ct value of the CY5 fluorescent channel is ≤30, it indicates that Escherichia coli is positive;

[0026] When the Ct value of the FAM fluorescence channel is ≤25 or the Ct value of the VIC or ROX fluorescence channels is ≤30, and the melting curve does not produce a specific peak, the sample needs to be re-extracted and tested. If the retest result is still the same, it is interpreted as negative.

[0027] The beneficial effects of this invention: This invention utilizes multiplex probe melting curve technology, employing four probes with different fluorescence levels combined with multiplex melting curves to develop a detection kit for eight common bacterial targets in bloodstream infections. This invention possesses the sensitivity and specificity of quantitative real-time PCR, and offers higher throughput, simpler operation, and lower cost compared to quantitative real-time PCR. It provides a reliable experimental method for the clinical diagnosis of bloodstream infections. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0029] Figure 1 Melting curves and amplification curves for Gram-positive results.

[0030] Figure 2 Melting curves and amplification curves for Gram-negative results.

[0031] Figure 3 Melting and amplification curves of the human genome.

[0032] Figure 4 The result for Klebsiella pneumoniae in the VIC channel was positive.

[0033] Figure 5 The result for Staphylococcus aureus in the VIC channel was positive.

[0034] Figure 6 The results for Streptococcus pneumoniae in the ROX channel were positive.

[0035] Figure 7 The results for Streptococcus agalactiae in the ROX channel were positive.

[0036] Figure 8 Enterococci showed a positive result in the ROX channel.

[0037] Figure 9 The result was positive for Escherichia coli in the CY5 channel. Detailed Implementation

[0038] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0039] Example 1:

[0040] A kit for detecting bloodstream bacterial nucleic acid using a multiplex probe melting curve method includes a fluorescent PCR reaction solution, Taq DNA polymerase solution, a positive control, and a negative control. The fluorescent PCR reaction solution contains PCR buffer (TE buffer, pH = 8.2), MgCl2, dNTPs, upstream primers for eight bacterial targets, downstream primers for eight bacterial targets, and fluorescent probes for eight bacterial targets. The eight bacterial targets are Gram-negative bacteria, Gram-positive bacteria, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae, and Enterococcus. The upstream primer sequences, downstream primer sequences, and fluorescent probe sequences for the eight bacterial targets are shown in Table 1.

[0041] The concentrations of each component and raw material in the kit, as well as the concentration of the PCR reaction, are as follows:

[0042] Component name (primer and probe) Raw material concentration 1 serving volume Reaction concentration SEQ ID NO 1 100 uM 0.03 μL 120 nM SEQ ID NO 2 100 uM 0.1 μL 400 nM SEQ ID NO 3 100 uM 0.1 μL 400 nM SEQ ID NO 4 100 uM 0.1 μL 400 nM

[0043]

[0044] Table 1 lists the upstream primer sequences, downstream primer sequences, and fluorescent probe sequences corresponding to the eight bacterial targets.

[0045]

[0046]

[0047] The positive control in the kit of this invention is a mixed solution of 8 bacterial target amplification sequences, dissolved in TE buffer. The 8 bacterial target amplification sequences are shown in Table 2.

[0048] Table 2 Amplification sequences of bloodstream infection bacteria

[0049]

[0050]

[0051] The negative control in this kit is sterile water for injection, and the enzyme solution is Taq DNA polymerase. This kit should be stored at -20°C.

[0052] This kit also includes a box and a lid. The box has eight wells for holding two fluorescent PCR reaction tubes, two enzyme solution tubes, two positive control tubes, and two negative control tubes. The fluorescent PCR reaction tubes contain the fluorescent PCR reaction solution, the enzyme solution tubes contain the enzyme solution, the positive control tubes contain the positive control, and the negative control tubes contain the negative control. The eight wells are designated for placing the fluorescent PCR reaction tubes, enzyme solution tubes, positive control tubes, and negative control tubes, respectively. The lid contains the instruction manual.

[0053] Example 2:

[0054] The method for detecting bloodstream bacterial nucleic acid using the kit described in Example 1 includes the following steps:

[0055] (1) Nucleic acid extraction: Take 0.5 mL of serum sample from children suspected of having sepsis in clinical practice and put it into an EP tube. Extract the nucleic acid from the serum sample using magnetic bead extraction or column extraction.

[0056] (2) Reagent preparation: Take 19 μL of reaction solution and 1 μL of enzyme solution to prepare 20 μL of PCR qualitative reaction solution; the main components of the reaction solution include primers, probes, magnesium chloride, and dNTPs, wherein the concentration of magnesium chloride is 4 mM and the concentration of dNTPs is 10 mM; the enzyme solution is Taq DNA polymerase with an enzyme activity of 5 U;

[0057] (3) Amplification and detection: Take 5 μL of the extraction product obtained in step (1), 5 μL of the positive control, and 5 μL of the negative control, and add them to 20 μL of the PCR quantitative reaction liquid obtained in step (2) above to prepare a total PCR system of 25 μL. Detect the PCR system using a fluorescence quantitative PCR instrument with 4 or more colors. The specific reaction procedure is as follows (taking the domestic Hongshi SLAN-96P fluorescence quantitative PCR instrument as an example), as shown in Table 3:

[0058] Table 3 Reaction Program Settings SLAN-96P

[0059]

[0060] (4) Results Analysis

[0061] Analysis of test results as follows Figure 1 As shown:

[0062] Positive cutoff value: This invention detects critical positive and negative samples, uses statistical methods to plot ROC curves, and employs the Youden index to determine the positive cutoff value.

[0063] A negative specimen is one in which the CT values ​​of the FAM probe are all greater than 25, the CT values ​​of the VIC probe are all greater than 30, the CT values ​​of the ROX probe are all greater than 30, and the CT value of the CY5 probe is all greater than 30.

[0064] When the Ct value of the FAM fluorescence channel is ≤25, it indicates that the bacteria are either Gram-negative or Gram-positive. Further analysis of the melting curve shows that a melting peak at 69.0±1℃ indicates Gram-positive bacteria, and a melting peak at 76.0±1℃ indicates Gram-negative bacteria.

[0065] When the Ct value of the VIC fluorescence channel is ≤30, it indicates Staphylococcus aureus or Klebsiella pneumoniae. Further analysis of the melting curve shows that a melting peak at 72.0±1℃ indicates Staphylococcus aureus positivity, and a melting peak at 63±1℃ indicates Klebsiella pneumoniae positivity.

[0066] When the Ct value of the ROX fluorescence channel is ≤35, it indicates that the bacteria are positive for Streptococcus pneumoniae, Streptococcus agalactiae, and Enterococcus. Further analysis of the melting curve shows that a melting peak at 66.0±1℃ indicates a positive result for Streptococcus agalactiae, a melting peak at 69.0±1℃ indicates a positive result for Streptococcus pneumoniae, and a melting peak at 77.0±1℃ indicates a positive result for Enterococcus.

[0067] When the Ct value of the CY5 fluorescent channel is ≤30, it indicates that Escherichia coli is positive;

[0068] When the Ct value of the FAM fluorescence channel is ≤25 or the Ct value of the VIC or ROX fluorescence channels is ≤30, and the melting curve does not produce a specific peak, the sample needs to be re-extracted and tested. If the retest result is still the same, it is interpreted as negative.

[0069] Validation of the kit for clinical samples: Positive samples of common bloodstream infection bacteria in clinical practice were selected, and DNA was extracted and then validated using the kit of this invention.

[0070] The selected samples included *Streptococcus agalactiae*, *Pseudomonas aeruginosa*, *Streptococcus pneumoniae*, *Acinetobacter baumannii*, *Klebsiella pneumoniae*, *Staphylococcus epidermidis*, *Enterococcus*, *Escherichia coli*, *Streptococcus gallolyticus*, *Staphylococcus aureus*, *Streptococcus maltophilia*, Salmonella, *Stenotrophomonas maltophilia*, and human genome samples. *Streptococcus agalactiae*, *Streptococcus pneumoniae*, *Staphylococcus epidermidis*, *Enterococcus*, *Streptococcus gallolyticus*, *Staphylococcus aureus*, and *Streptococcus maltophilia* showed Gram-positive results in the FAM channel. Figure 1 Salmonella, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli showed Gram-negative results in the FAM channel. Figure 2 ).

[0071] The human genome shows a Gram-negative result. Figure 3Klebsiella pneumoniae showed a positive result in the VIC channel. Figure 4 Staphylococcus aureus showed a positive result in the VIC channel. Figure 5 Streptococcus pneumoniae showed a positive result in the ROX channel. Figure 6 Streptococcus agalactiae showed a positive result in the ROX channel. Figure 7 Enterococci showed a positive result in the ROX channel. Figure 8 Escherichia coli showed a positive result in the CY5 channel. Figure 9 ).

[0072] This invention detects 31 clinical samples from Zhejiang University Children's Hospital, including 2 samples of Klebsiella pneumoniae, 3 samples of Staphylococcus aureus, 3 samples of Streptococcus pneumoniae, 3 samples of Streptococcus agalactiae, 3 samples of Enterococcus, 3 samples of Escherichia coli, and 14 other negative samples. The detection accuracy rate is 100%.

[0073] The positive standard of this invention is used to detect Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae, and Enterococcus, which are diluted to 100,000 copies / ml, 50,000 copies / ml, 20,000 copies / ml, 10,000 copies / ml, 8,000 copies / ml, 5,000 copies / ml, and 1,000 copies / ml, respectively. The detection results show that at a concentration of 10,000 copies / ml, the detection rate of Klebsiella pneumoniae and Enterococcus pneumoniae is 95% (19 / 20), and the detection rate of the other bacteria is 100% (20 / 20). Therefore, the minimum detection concentration of this invention is determined to be 10,000 copies / ml.

[0074] This invention utilizes a single reaction tube to detect four fluorescence channels (FAM, VIC, ROX, and CY5 channels) and melting curves, enabling the detection of eight bacterial targets (Gram-negative bacteria, Gram-positive bacteria, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae, and Enterococcus), significantly increasing the throughput of single-tube reactions while requiring only the cost of single-tube reagents. The detection reaction can be performed on a standard quantitative PCR instrument, offering simple result interpretation, eliminating the risk of contamination without opening the tube, and making it suitable for all clinical PCR laboratories in medical institutions. Furthermore, this detection technology combines quantitative PCR and melting curve analysis, achieving the same high sensitivity and specificity as quantitative PCR.

[0075] This invention utilizes multiplex probe melting curve technology, employing four probes in conjunction with fluorescent PCR to develop a detection kit for nucleic acids targeting eight common bloodstream bacteria. This invention possesses the sensitivity and specificity of quantitative real-time PCR, but offers higher throughput, simpler operation, and lower cost compared to quantitative real-time PCR. It provides a reliable experimental method for the clinical diagnosis of bloodstream infections.

[0076] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A kit for detecting bacterial nucleic acid of bloodstream infection using multiplex probe melting curve method, characterized by comprising: The kit comprises a fluorescent PCR reaction solution, a DNA polymerase solution, the fluorescent PCR reaction solution containing a PCR buffer solution, magnesium chloride, dNTPs, an upstream primer, a downstream primer, and eight bacterial target corresponding fluorescent probes, the eight bacterial targets being Gram-negative bacteria, Gram-positive bacteria, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus agalactiae, and Enterococcus; ​ The sequence of the upstream primer is shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 20; The sequence of the downstream primer is shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, or SEQ ID NO: 21; The sequence of the eight bacterial target corresponding fluorescent probes is shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 22; The fluorescent labeling group of SEQ ID NO: 3 and SEQ ID NO: 4 is FAM, the fluorescent labeling group of SEQ ID NO: 7 and SEQ ID NO: 10 is VIC, the fluorescent labeling group of SEQ ID NO: 13 is CY5, and the fluorescent labeling group of SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22 is ROX.

2. The kit for detecting bacterial nucleic acid of bloodstream infection according to claim 1, wherein: The kit further comprises a positive control and a negative control, the positive control being a mixture of the amplification sequences of the eight bacterial targets dissolved in a TE buffer solution, and the negative control being sterile water.

3. The kit for detecting bacterial nucleic acid of blood stream infection by multiplex probe melting curve method according to claim 1 or 2, characterized in that: The DNA polymerase solution is Taq DNA polymerase. The PCR buffer solution is a TE buffer solution with a pH of 8.2.

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