Compositions, kits, methods of detecting different bordetella and uses thereof

Abstract

The present application belongs to the field of molecular biology detection, and particularly relates to detection of pathogenic bacteria related to respiratory tract infection, and more particularly to detection of Bordetella parapertussis, Bordetella hockingense and Bordetella bronchiseptica. The composition for combined detection provided by the present application mainly utilizes a multiplex fluorescence PCR analysis method, and different pathogenic bacteria are detected by detecting target points on different pathogenic bacteria, so that detection and differentiation of Bordetella parapertussis, Bordetella hockingense and Bordetella bronchiseptica are simultaneously achieved in a single tube reaction system. The composition of the present application has higher 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 pathogens related to respiratory tract infections, and more specifically, it relates to the detection of Bordetella parapertussis, Bordetella hominis, and Bordetella bronchiseptica. Background Technology

[0002] Bordetella parapertussis is a Gram-negative bacterial pathogen that colonizes the respiratory tract and causes pertussis (whooping cough). However, the severity of illness caused by Bordetella parapertussis is considered to be shorter and milder than that caused by Bordetella pertussis, the predominant causative agent of pertussis in humans. Comparative genomic analysis of Bordetella species indicates that both Bordetella pertussis and Bordetella parapertussis evolved independently from a bronchosepticemic ancestor. Bordetella parapertussis has diverged into two distinct lineages: one causing pertussis in infants, and the other infecting sheep.

[0003] *Bordetella hovellii* is a Gram-negative, rod-shaped (or primarily coccidia and short rod-shaped), slow-growing microorganism. It is believed to cause invasive infections (bacteremia, meningitis, endocarditis, pericarditis, pneumonia, and arthritis) and pertussis symptoms. Because the standard diagnostic test for pertussis is not species-specific, *Bordetella hovellii* infection is often misdiagnosed as *Bordetella pertussis* infection. Antibiotic treatment can also present problems, as *Bordetella hovellii* is less sensitive than expected to macrolides (empirically used to treat pertussis) and third-generation cephalosporins (commonly used to treat invasive infections). *Bordetella hovellii* continues to adapt to humans, and its virulence may increase, thus requiring better diagnostic methods and epidemiological surveillance.

[0004] Cultured *Baudolinium cholerae* via biological culture (blood, pleural, or joint cultures, or nasopharyngeal samples) is cumbersome, with a mean incubation time of 40 hours for positive blood cultures, and poor growth on MacConkey agar. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized bacterial identification by providing rapid and accurate identification of most pathogens obtained in clinical microbiology. For pathogens that are difficult to identify accurately using biochemical methods, such as *Baudolinium cholerae*, MALDI-TOF MS is an ideal identification instrument. However, only a few published works report on *Baudolinium cholerae* with MALDI-TOF MS. The gold standard for identification remains 16S rDNA sequencing. However, sequences obtained by 16S sequencing are often very similar to those obtained from *Bordetella pertussis*, necessitating further sequence analysis, such as PCR detection of *Baudolinium cholerae*-specific genes.

[0005] *Bordetella bronchiseptica* is a small, Gram-negative cocci in the genus *Bordetella*. Unlike the picky *Bordetella pertussis*, *Bordetella bronchiseptica* grows readily on standard bacterial media. *Bordetella bronchiseptica* exhibits a predisposition to respiratory infections in animals such as dogs, cats, rabbits, pigs, horses, and mice. It causes tracheobronchitis or "kennel cough" in dogs, atrophic rhinitis in pigs, and nasal congestion in rabbits. This predisposition makes *Bordetella bronchiseptica* infection uncommon in humans, and because animals are infected before most humans, it is considered a zoonotic disease. *Bordetella bronchiseptica* is an obligate aerobe, growing for 1 to 2 days on standard laboratory media such as blood, chocolate, and MacConkey agar. Identification is performed using biochemical and phenotypic methods or molecular methods such as polymerase chain reaction (PCR), 16S rRNA sequencing, and matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF). This pathogen may cross-react with antibodies against *Bordetella pertussis*, leading to false positives in *Bordetella pertussis* fluorescent antibody tests. Although the detection of Bordetella pertussis has long been considered specific, the target insertion gene sequence IS481 used in current PCR detection may be present in strains of Bordetella bronchiseptica, raising concerns about false identification.

[0006] Therefore, there is a need in the field for a product that can easily and quickly detect the above-mentioned pathogens, and 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 pathogens, comprising:

[0008] The upstream primer, downstream primer, and probe for detecting Bordetella parapertussis are shown in SEQ ID NO:1-3;

[0009] For example, the upstream primers, downstream primers, and probes for detecting Bordetella hoobini, as shown in SEQ ID NO:4–6; and

[0010] The upstream primer, downstream primer, and probe for detecting Bordetella bronchiseptica are shown in SEQ ID NO:7–9.

[0011] Bordetella parapertussis, Bordetella hominis, and Bordetella bronchiseptica have all been reported to infect humans. Due to their high genetic similarity, it is difficult to distinguish them using 16S rDNA sequencing, the gold standard for routine clinical diagnosis and species identification.

[0012] 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 *Bordetella pertussis*, *Bordetella cholerae*, and *Bordetella bronchiseptica* in a single-tube reaction system. The composition of this invention exhibits higher detection sensitivity (up to 500 copies / mL), better specificity, and more accurate detection.

[0013] Furthermore, the composition includes an upstream primer, a downstream primer, and a probe for detecting an internal standard.

[0014] In some specific implementations, the internal control is a human internal control gene. In one specific implementation, the internal control is RNase P.

[0015] In some specific embodiments, the composition further includes upstream primers, downstream primers, and probes for detecting internal standards, as shown in SEQ ID NO:10-12.

[0016] 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.

[0017] 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.

[0018] In some specific implementation schemes, the fluorescent reporter group of the Bordetella parapertussis probe is FAM; the fluorescent reporter group of the Bordetella bronchiseptica probe is HEX (or VIC); the fluorescent reporter group of the Bordetella hopnea probe is ROX; and the fluorescent reporter group of the internal standard probe is CY5.

[0019] 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.

[0020] The compositions of this invention can be arbitrarily combined to detect any combination of four 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.

[0021] For example, it may include any 3 pairs of the above 4 pairs of primers and probes, any 2 pairs of the above 4 pairs of primers and probes, or any 1 pair of the above 4 pairs of primers and probes.

[0022] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0023] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0024] Furthermore, the 3' end of the probe also has a quenching group, such as BHQ1 or BHQ2.

[0025] In one specific implementation, the 3' end of the probe is BHQ1.

[0026] In one specific embodiment, each component of the composition of the present invention is contained in a separate package.

[0027] In one specific embodiment, the components of the composition of the present invention are contained in the same package.

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

[0029] 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 pathogens, wherein the pathogens are Bordetella parapertussis, Bordetella hominis, and Bordetella bronchiseptica.

[0030] Thirdly, the present invention provides a kit for the combined detection and differentiation of pathogens, the kit comprising the composition of the present invention as described above.

[0031] Furthermore, the kit also includes negative and positive controls.

[0032] In one specific implementation, the negative control is at least one of DEPC H2O, physiological saline, and an internal standard gene. The positive control is at least one of a fragment plasmid or fragment DNA of Bordetella parapertussis, Bordetella hominis, or Bordetella bronchiseptica.

[0033] Furthermore, the kit also includes dNTPs, PCR buffer, and Mg. 2+ At least one of them.

[0034] Furthermore, the kit also includes at least one of the following: a nucleic acid release reagent, a nucleic acid extraction reagent, and a DNA polymerase.

[0035] Furthermore, the kit also includes nucleic acid release reagents, nucleic acid extraction reagents, dNTPs, dUTPs, uracil glycosylase (UDG), DNA polymerase, PCR buffer, and Mg... 2+ At least one of them.

[0036] Furthermore, the concentration of the DNA polymerase is 3 U / reaction to 15 U / reaction, for example, the DNA polymerase can be Taq polymerase.

[0037] In one specific embodiment, the kit of the present invention includes Taq enzyme, Mg 2+ dNTPs (U), primers, probes, and PCR buffer.

[0038] 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.

[0039] 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.

[0040] Fourthly, a method for the joint detection and differentiation of pathogens is provided, the method comprising the following steps:

[0041] 1) Extract or release nucleic acid from the sample to be tested;

[0042] 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;

[0043] 3) Obtain and analyze the results.

[0044] In this invention, the sample used for detection can be a nasal swab, a throat swab, etc., but is not limited to these.

[0045] Furthermore, the reaction conditions for the quantitative real-time PCR are as follows:

[0046] UDG enzyme reaction: temperature 40–60℃, time 30–150 seconds, 1 cycle; cDNA pre-denaturation: temperature 94℃, time 3–6 min, 1 cycle; denaturation: temperature 94℃, time 5–20 seconds; annealing: temperature 55℃–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0047] In one specific implementation, a method is provided for the combined detection and differentiation of pathogens for non-diagnostic purposes, the method comprising the following steps:

[0048] 1) Extract or release nucleic acid from the sample to be tested;

[0049] 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;

[0050] 3) Obtain and analyze the results.

[0051] Furthermore, the reaction conditions for the quantitative real-time PCR are as follows:

[0052] UDG enzyme reaction: temperature 40–60℃, time 30–150 seconds, 1 cycle; cDNA pre-denaturation: temperature 94℃, time 3–6 min, 1 cycle; denaturation: temperature 94℃, time 5–20 seconds; annealing: temperature 55–60℃, time 10–60 seconds, 30–50 cycles; fluorescence collection.

[0053] In this article, the term "non-diagnostic purpose" refers to a method not intended to obtain information about whether an individual is infected with the aforementioned pathogens and suffers from respiratory disease. For example, the method may be used to detect the presence of the aforementioned pathogens in a test culture (e.g., a throat swab). Attached Figure Description

[0054] Figures 1-4 The images show the detection results of the compositions of the present invention (representing Bordetella parapertussis, Bordetella hominis, Bordetella bronchiseptica, and internal standard, respectively).

[0055] Figures 5-7 The graph shows the sensitivity results of the compositions of the present invention (Bordeia parapertussis, Bordetella hominis, and Bordetella bronchiseptica, respectively).

[0056] Figure 8 This is a graph showing the specificity of the composition of the present invention;

[0057] Figures 9-11 The graph shows the precision results of the compositions of the present invention (Bordeia parapertussis, Bordetella hominis, and Bordetella bronchiseptica, respectively).

[0058] Figures 12-14 The images show the detection results of the comparative compositions of this invention (Bordeia parapertussis, Bordetella hominis, and Bordetella bronchiseptica, respectively). Detailed Implementation

[0059] 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.

[0060] Example 1: Primers and probes used in this invention

[0061] The primers and probes used in this invention are shown in Table 1 below:

[0062]

[0063]

[0064] Among them, the fluorescent reporter group of BPP is FAM; the fluorescent reporter group of BH is HEX (or VIC); the fluorescent reporter group of BBP is ROX; and the fluorescent reporter group of the internal standard probe is CY5.

[0065] Example 2: Method for detecting pathogens

[0066] The sample used in this invention is a nasopharyngeal swab. Viral nucleic acid is extracted using the magnetic bead method, and the following procedures are performed in the sample processing room:

[0067] 2.1 Take several 1.5 mL centrifuge tubes according to the number of samples to be tested, and add 300 μL of sample to each tube;

[0068] 2.2 Add 500 μL of extraction solution 1 and 50 μL of proteinase K-magnetic bead mixture; cap the tube, shake to mix for 30 s, and heat at 60 °C for 10 min.

[0069] 2.3 Let stand at room temperature for 1 minute, then centrifuge briefly at low speed. Place the centrifuge tube on a magnetic separator and slowly aspirate the waste liquid after 5 minutes (*Be careful not to touch the magnetic beads adsorbed on the inner wall of the tube).

[0070] 2.4 Add 200 μL of washing solution 1 and 600 μL of washing solution 2, vortex to mix for 30 seconds, centrifuge briefly at low speed, and then place the centrifuge tube in a magnetic separator. Magnetically aspirate for 3 minutes to completely remove and discard the liquid.

[0071] 2.6 Place the centrifuge tubes in the centrifuge and centrifuge briefly at low speed. Then place the centrifuge tubes back into the magnetic separator. Magnetize for 3 minutes to completely remove the liquid from the bottom of the tubes.

[0072] 2.7 Add 30-100 μL of elution buffer S (80 μL is recommended), vortex and mix for 30 seconds to wash the magnetic beads off the centrifuge tube wall to the bottom of the tube, and let stand at room temperature for 3 minutes; centrifuge briefly at low speed, place the centrifuge tube on the magnetic separator again for 3 minutes, and then transfer the eluted nucleic acid to a clean 1.5 mL centrifuge tube.

[0073] 2.8 Collect the above-processed samples, BPP / BH / BBP-positive controls, and BPP / BH / BBP-negative samples.

[0074] Add 10 μL of each control to the corresponding 0.2 mL PCR reaction tube, add 40 μL of PCR mixture to each tube, and cap the tubes.

[0075] The real-time fluorescence PCR reaction system is prepared as follows:

[0076] template 5-10μL <![CDATA[Mg 2+ ]]> 4-6mM dNTPs (100mM) 0.2-0.4mM Taq enzyme (5 U / μl) 5-15U Primers 100-500nM probe 50-250nM PCR buffer (1x) Add to 50μL

[0077] The PCR amplification program should be set up as follows:

[0078]

[0079] Results analysis:

[0080] 1) The target detection signals are FAM, HEX (or VIC), and ROX; the human gene internal standard detection signal is CY5.

[0081] 2) Baseline settings: The baseline is generally set to 3-15 cycles, which can be adjusted according to the actual situation. The adjustment principle is: select a region where the fluorescence signal is relatively stable before exponential amplification; avoid signal fluctuations at the beginning of fluorescence acquisition; and reduce the endpoint (Ct) by 1-2 cycles compared to the earliest sample showing exponential amplification. Threshold settings: The principle is to set the threshold line so that it just exceeds the highest point of the normal negative control.

[0082] 3) Result Interpretation

[0083] A) For samples where a typical S-type amplification curve is detected in the FAM channel and Ct≤40, the report is BPP positive; for samples where a typical S-type amplification curve is not detected in the FAM channel, or Ct>40, and an amplification curve is present in the CY5 channel and Ct≤40, the report is BPP negative.

[0084] B) For samples where a typical S-type amplification curve is detected in the HEX channel and Ct≤40, the report is BH positive; for samples where a typical S-type amplification curve is not detected in the HEX channel, or Ct>40, and an amplification curve is present in the CY5 channel and Ct≤40, the report is BH negative.

[0085] C) For samples where a typical S-type amplification curve is detected in the ROX channel and Ct≤40, the sample is reported as BBP positive; for samples where a typical S-type amplification curve is not detected in the ROX channel, or Ct>40, but an amplification curve is present in the CY5 channel and Ct≤40, the sample is reported as BBP negative.

[0086] D) If no typical S-type amplification curve (No Ct value) is detected in the FAM, ROX, HEX, and CY5 channels, or if Ct > 40, it indicates that the cell content of the sample is too low or there is an interfering substance inhibiting the reaction. The test result of this sample is invalid. The cause should be found and eliminated, and the sample should be resampled and repeated.

[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 Bordetella parapertussis, Bordetella hominis, and Bordetella bronchiseptica on a Hongshi real-time PCR instrument, following the method in Example 2. The detection results are as follows: Figures 1-4 As shown in the figure, the compositions of the present invention can effectively detect various pathogens.

[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 various targets at concentrations of 5000, 1000, 500, and 200 copies / ml to simulate clinical samples, and multiplex PCR detection was performed on a Hongshi real-time PCR instrument. The results are as follows: Figures 5-7 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 this invention is 500 copies / mL.

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

[0092] The composition of this invention showed no cross-reactivity with common respiratory pathogens and other pathogens with similar infection symptoms (such as Bordetella pertussis, respiratory syncytial virus, respiratory adenovirus, influenza A virus, influenza B virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, Epstein-Barr virus, rhinovirus, human metapneumovirus, Acinetobacter baumannii, Escherichia coli, Serratia marcescens, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, Legionella pneumophila, etc.). Results are as follows: Figure 8 As shown.

[0093] Example 6: Precision of the composition of the present invention

[0094] Two quality control samples, one with strong positive and one with weak positive (10,000 copies / ml and 2,000 copies / ul, respectively), were selected for intra-assay and inter-assay precision determination. Each sample was measured 10 times. The results showed that the detection rate of both strong and weak positive reference samples was 100%, and the coefficient of variation (CV) of the intra-assay and inter-assay detection Ct values ​​was less than 5%, indicating that this kit has good intra-assay and inter-assay precision. Figures 9-11 As shown.

[0095] Comparative Example 1: Other primers and probes designed in this invention that do not perform well.

[0096] 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.

[0097] Therefore, the inventors also designed other primers and probes to form different detection systems 1-3 (sequences not shown), which were also used to detect the aforementioned pathogens. Specific detection results are as follows: Figures 12-14 As shown in the figure, only some amplification curves were detected, and the amplification curves showed low increases and no plateau phase. Other targets did not even show any amplification curves. Therefore, the overall detection effect was poor.

Claims

1. A composition for the joint detection and differentiation of pathogens, comprising: The upstream primer, downstream primer, and probe for detecting Bordetella parapertussis are shown in SEQ ID NO:1~3; The upstream primer, downstream primer, and probe for detecting Bordetella hominis are shown in SEQ ID NO:4~6; as well as The upstream primer, downstream primer, and probe for detecting Bordetella bronchiseptica 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 composition further includes upstream primers, downstream primers, and probes for detecting internal standards, as shown in SEQ ID NO:10~12.

4. The composition according to claim 3, characterized in that, The fluorescent reporter group for Bordetella parapertussis is FAM; the fluorescent reporter group for Bordetella hopnea is HEX or IVC; the fluorescent reporter group for Bordetella bronchiseptica is ROX; and the fluorescent reporter group for the internal standard probe is CY5.

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 pathogens, wherein, The pathogens are Bordetella parapertussis, Bordetella hominis, and Bordetella bronchiseptica.

7. A kit for detecting and differentiating 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 release reagent, nucleic acid extraction reagent, DNA polymerase, dNTPs, dUTPs, UDG enzyme, PCR buffer, and Mg. 2+ At least one of them.

10. A method for detecting and differentiating pathogens for non-diagnostic purposes, characterized in that, The pathogens are Bordetella parapertussis, Bordetella hominis, and Bordetella bronchiseptica. The method includes the following steps: 1) Extract or release 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.

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