CN113088577A - Method and kit for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof - Google Patents
Method and kit for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof Download PDFInfo
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Abstract
The invention discloses a fluorescence PCR detection method for simultaneously detecting Bordetella pertussis DNA and drug-resistant mutation sites thereof, which comprises the steps of adding a sample DNA to be detected and an internal standard into a fluorescence PCR reaction solution for amplification reaction, judging a reaction result according to a Ct value of a fluorescence signal detection channel, and distinguishing whether a sample to be detected is Bordetella pertussis or drug-resistant Bordetella pertussis, thereby guiding the clinical correct use of antibiotics and preventing the abuse of the antibiotics. The invention also provides a kit for the detection method of the bordetella pertussis and the drug-resistant mutation sites thereof, wherein the kit comprises a fluorescent PCR reaction solution and an internal standard for detecting the bordetella pertussis. The method and the kit can save detection time and cost, and have the advantages of high detection accuracy, high sensitivity, good specificity and good precision.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a method and a kit for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof.
Background
Whooping cough is a severe acute respiratory infectious disease caused by bordetella pertussis, which is characterized by a typical spasmodic cough accompanied by a roommar-like echo. The widespread vaccination of pertussis, although causing a significant reduction in the incidence of the disease, is to be seen in infants less than 6 months of age, who are still weak against pertussis or who are not as age-matched because the antibodies generated by vaccination decrease with age and the antibodies in pregnant women are rarely transmitted to the fetus. In addition, the antibody generated by vaccination declines with age, and the occurrence and propagation of drug-resistant bordetella pertussis strains and the like lead to the gradual increase of the number of people suffering from pertussis among vaccinated adolescents and adults, and the outbreak or epidemic occurs in local areas, and the occurrence and propagation of drug-resistant bordetella pertussis strains form a pertussis reappearance phenomenon, so that people face a new challenge in the prevention and control of pertussis. It is noteworthy that the number of vaccinated adolescents and adults suffering from pertussis is increasing, the clinical symptoms are not typical, are often overlooked, and can be a source of infection threatening the health of young children. Therefore, it is important to enhance monitoring of pertussis and drug resistance.
Macrolide antibiotics represented by erythromycin are the first choice antibiotics for treating pertussis and for preventive medication, such as erythromycin, azithromycin, roxithromycin or clarithromycin. The curative effect is related to the morning and evening of medication, the application of the antibiotic in catarrh stage can relieve or even prevent the occurrence of the pertussis, and the application after the pertussis stage can not shorten the clinical process of the pertussis, but can shorten the bacteria-expelling stage and prevent secondary infection. However, the first erythromycin resistant strain of Bordetella pertussis was discovered in 1994 worldwide in the United states and has attracted widespread attention. With the increasing severity of the drug resistance problem of various bacteria in the world, the phenomenon of macrolide antibiotic resistance of bordetella pertussis also appears in China. For better guidance of clinical medication, detection of bordetella pertussis nucleic acid and detection of drug-resistant mutation sites may purposefully use different therapeutic drugs for pertussis patients and drug-resistant patients.
The traditional laboratory detection method of bordetella pertussis mainly comprises bacterial culture, serological test, conventional molecular biological method and the like. The bacteria culture method is used as a gold standard, has high specificity, but has complex culture technology and sensitivity influenced by various factors such as antibacterial drug use, disease course, specimen transport conditions, specimen quality, culture operation methods and the like, so that the sensitivity is low, the positive rate is low, the time consumption is long, and the method is not suitable for rapid diagnosis of early pathogen infection.
At present, the pertussis etiology detection method in the market is mainly an enzyme-linked immunosorbent assay, a Pertussis Toxin (PT) antibody is detected, a single serum is usually detected by a PT-IgG antibody, but the age and the immune state of an infant patient are fully considered; in addition, 2 serum samples are taken from suspected patients in the early onset and recovery stages, and the PT-IgG titer is remarkably increased (> 2-4 times) to determine whether the pertussis bacillus is infected. However, the specificity IgG is positive only for indicating that the corresponding bacterial infection exists, the kit cannot be used for early infection judgment and curative effect monitoring, the detection positive rate is high only after the disease course is about 10 days, the sampling time is long, and the commercialized kit lacks of standardized judgment indexes, which are not favorable for early diagnosis and rapid diagnosis of Bordetella pertussis. In addition, false negative can also occur due to weak immune response, which has certain limitation on some children patients with immunodeficiency and immune system which is not developed completely; in addition, bacterial specific antibody detection can only detect bordetella pertussis infection and cannot distinguish between drug resistance and drug resistance.
Real-time PCR, also known as fluorescence quantitative PCR, is a novel nucleic acid quantitative technique introduced by Applied Biosystems in the United states in 1996, and is widely used with the introduction of fluorescence PCR instruments. The technology is characterized in that a fluorescence resonance energy transfer phenomenon is applied on the basis of conventional PCR, a fluorescence labeled probe is added, nucleic acid amplification, hybridization, spectral analysis and real-time detection technologies are ingeniously combined together, the quantity of a specific product is measured in time by continuously monitoring the intensity of a fluorescence signal during PCR exponential amplification, and the initial quantity of a target gene is deduced according to the quantity, so that a specific detection result can be quickly obtained.
The research shows that the pertussis erythromycin resistance phenomenon is related to a nucleotide site mutation in the V region of 23S rRNA gene of genome, namely A2047G. Several reports follow that confirm: in all of bordetella pertussis resistant to erythromycin or macrolide antibiotics, a2047G mutation in the 23S rRNA gene occurs. Currently, single-base mutation can be mostly detected only by a melting curve of fluorescence quantitative PCR or a sequencing method, and the result is determined by respectively using Tm value detection (generally, Tm values of wild type and mutant type are different by 0.24 ℃) which is the influence of single-base mutation on melting temperature, but the requirements on instruments and operators are high; the sequencing method needs to open the cover of the amplified product, perform restriction fragment enzyme digestion electrophoresis observation, judge whether mutation occurs according to the size of the strip, or perform sequencing of the first generation and compare the sequence with a standard strain to judge whether mutation occurs, so the method is easy to pollute the laboratory. Therefore, the detection of a single base mutation in the 23S rRNA variable region a2047G of bordetella pertussis resistance region remains a technical difficulty.
In the prior art, CN 103667512a discloses a primer and a detection method for rapidly detecting drug resistance of bordetella pertussis erythromycin from a specimen, which cannot directly determine whether bordetella pertussis is mutated or not, and first determine whether the bordetella pertussis erythromycin is mutated by other methods, and on the basis, primers are designed for variable regions on both sides of 2047 locus included in 23S rRNA gene of bordetella pertussis, so as to detect drug resistance of bordetella pertussis erythromycin. Although the method is designed aiming at the pertussis drug-resistant 23s rRNA region, the method adopts a sequencing method, needs to be subjected to common PCR amplification, then is subjected to a generation sequencing method, and finally is compared with a standard strain sequence according to a sequencing peak diagram to judge whether mutation occurs.
Disclosure of Invention
In order to solve the existing problems, the invention provides a fluorescence PCR detection system for simultaneously detecting Bordetella pertussis DNA and drug-resistant mutation sites thereof, which can directly judge results in a totally-enclosed amplification system of the same reaction tube and distinguish whether a sample to be detected is Bordetella pertussis or drug-resistant Bordetella pertussis, thereby guiding the clinical correct use of antibiotics and preventing the abuse of antibiotics. The method saves detection time and cost, and has high detection accuracy, high sensitivity, good specificity and good precision.
The solution of the invention is as follows:
the invention relates to a method for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof, which comprises the following steps: adding the DNA and the internal standard of a sample to be detected into a fluorescent PCR reaction solution for amplification reaction, and then interpreting the reaction result according to the Ct value of a fluorescent signal detection channel, wherein the interpretation is carried out according to the following method:
(1) when the Ct value of the bordetella pertussis nucleic acid fluorescence signal detection channel is more than 26.00, the identification is carried out according to the following steps:
when a Ct value detected by an internal standard is less than or equal to 36.00, a fluorescence signal of a fluorescence signal detection channel of bordetella pertussis nucleic acid increases and is in a typical S-shaped curve, the Ct value is less than or equal to 36.00, and a fluorescence signal of the fluorescence signal detection channel of a bordetella pertussis drug-resistant mutation site has no obvious amplification curve, the DNA of the sample to be detected is bordetella pertussis DNA, but the A204 2047G drug-resistant mutation does not occur;
when the Ct value detected by an internal standard is less than or equal to 36.00, the fluorescence signals of the detection channels of the bordetella pertussis nucleic acid and the bordetella pertussis drug-resistant mutation site fluorescence signals are increased and are in typical S-shaped curves, and the Ct values are less than or equal to 36.00, the DNA of the sample to be detected is bordetella pertussis DNA, and A2047G drug-resistant mutation occurs;
when the Ct value detected by the internal standard is less than or equal to 36.00, the Ct value of a detection channel of a bordetella pertussis nucleic acid fluorescence signal is greater than 36.00 and a typical S-shaped curve does not exist, the DNA of the sample to be detected is not the bordetella pertussis DNA;
(2) when the Ct value of the bordetella pertussis nucleic acid fluorescence signal detection channel is less than or equal to 26.00, judging according to the following steps:
when the Ct value of an internal standard detection channel is less than or equal to 36.00, the fluorescence signal of a Bordetella pertussis nucleic acid fluorescence signal detection channel is increased and is in a typical S-shaped curve, and when the Ct value of the Bordetella pertussis nucleic acid fluorescence signal detection channel is less than or equal to 26.00, the Bordetella pertussis drug-resistant mutation site fluorescence signal detection channel has an amplification curve; then, whether the bordetella pertussis is mutated or not needs to be distinguished by calculating the Ct difference delta Ct between the bordetella pertussis nucleic acid fluorescence signal and the bordetella pertussis drug-resistant mutation site fluorescence signal:
when the delta Ct is less than or equal to 6, the DNA of the sample to be detected is Bordetella pertussis and A2047G drug-resistant mutation occurs;
and when the delta Ct is more than 6, the DNA of the sample to be detected is Bordetella pertussis and the A2047G drug resistance mutation does not occur.
Preferably, the fluorescent PCR reaction solution comprises the following components:
(1) a primer group: the concentration of each primer in the primer group is 0.6 mu M;
(2) and (3) probe group: the concentration of each probe in the probe group is 0.3 mu M;
(3) taq DNA polymerase: the concentration is 1.5U/reaction;
(4) reaction buffer: dNTP concentration 0.2mM, MgCl2The concentration was 2.5mM, the final concentration of KCl was 390mM, the pH of Tris-HCl was 9.39, and the final concentration was 250 mM.
Preferably, the primer set comprises: primer BP-F4: a forward primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.1 in a sequence table; primer BP-R4: a reverse primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.2 in a sequence table; primer MBP-F4: a forward primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.4 in a sequence table; primer MBP-R2: the reverse primer for detecting the bordetella pertussis drug-resistant mutation site is shown as a sequence in SEQ ID NO.5 in a sequence table.
Preferably, the set of probes comprises: probe BP-P2-FAM: a probe for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.3 in a sequence table; probe MBP-P2-VIC: a probe for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.6 in a sequence table; the bordetella pertussis nucleic acid fluorescence signal detection channel is a fluorescence detection channel of FAM in the probe BP-P2-FAM; the Bordetella pertussis drug-resistant mutation site fluorescence signal detection channel is a VIC fluorescence detection channel in the probe MBP-P2-VIC.
Preferably, the internal standard is a beta-globin gene (HBB), and the primer probes of the internal standard are: the forward primer beta-Globin-F1 is shown as SEQ ID NO.7 in the sequence table; the reverse primer beta-Globin-R2 is shown as SEQ ID NO.8 in the sequence table; the probe beta-Globin-CY 5 is shown as SEQ ID NO.9 in the sequence table.
Preferably, the amplification reaction conditions are: 94 ℃ for 2 min; entering a circulation stage: denaturation at 94 ℃ for 10s, annealing and extension at 55 ℃ for 35s, and reaction for 40 cycles.
The invention also provides a kit for the detection method of the bordetella pertussis and the drug-resistant mutation sites thereof, wherein the kit comprises a fluorescent PCR reaction solution and an internal standard for detecting the bordetella pertussis.
Preferably, the fluorescent PCR reaction solution comprises the following components:
(1) a primer group: the concentration of each primer in the primer group is 0.6 mu M; the primer group comprises: primer BP-F4: a forward primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.1 in a sequence table; primer BP-R4: a reverse primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.2 in a sequence table; primer MBP-F4: a forward primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.4 in a sequence table; primer MBP-R2: a reverse primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.5 in a sequence table;
(2) and (3) probe group: the concentration of each probe in the probe group is 0.3 mu M; the probe set comprises: probe BP-P2-FAM: a probe for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.3 in a sequence table; probe MBP-P2-VIC: a probe for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.6 in a sequence table;
(3) taq DNA polymerase: the concentration is 1.5U/reaction;
(4) reaction buffer: dNTP concentration 0.2mM, MgCl2The concentration is 2.5mM, the final concentration of KCl is 390mM, the pH of Tris-HCl is 9.39, and the final concentration is 250 mM;
(5) primer probes of the internal standard: the forward primer beta-Globin-F1 is shown as SEQ ID NO.7 in the sequence table; the reverse primer beta-Globin-R2 is shown as SEQ ID NO.8 in the sequence table; the probe beta-Globin-CY 5 is shown as SEQ ID NO.9 in the sequence table.
Preferably, the internal standard is a fragment of the beta-globin gene (HBB).
The invention has the beneficial effects that:
(1) can be in 2 hours while the specificity detect pertussis bacillus nucleic acid positive and drug resistance, save time and cost, and the degree of accuracy is high, and sensitivity is high, and the specificity is good, and precision is good: the method utilizes a fluorescent quantitative PCR method to carry out specific amplification, and then utilizes the difference value of amplification cycle numbers (Ct) corresponding to the amplification products of the sample to be detected when the fluorescent signals of different two channels reach a set fluorescent threshold value to distinguish whether the sample to be detected is the bordetella pertussis or drug-resistant bordetella pertussis, thereby not only being capable of specifically detecting the bordetella pertussis infection, but also being capable of guiding the clinical correct use of antibiotics and preventing the abuse of the antibiotics.
(2) Effectively prevent false positive results: the prior publications all use a promoter region as a target region of Bordetella pertussis, and since the region is relatively similar to the sequence of Bordetella parapertussis of other Bordetella, if not considered, false positive is easily caused.
Drawings
FIG. 1 is a graph of the amplification curve for the detection of positive control Bordetella pertussis and other pathogens in a specificity assay;
FIG. 2 is a graph showing the results of a sensitivity test for Bordetella pertussis at different gradient concentrations;
FIG. 3 is a graph showing the results of a sensitivity test for drug-resistant B.pertussis at different gradient concentrations;
FIG. 4 is 104A result chart of precision test of Bordetella pertussis at CFU/. mu.L concentration;
FIG. 5 is 103A result graph of precision test of CFU/. mu.l concentration of drug-resistant bordetella pertussis;
FIG. 6 is a diagram showing the results of an optimization experiment of the primer probe in the present detection method.
Detailed Description
The invention will be further illustrated by the following detailed description of specific embodiments, which are not to be construed as limiting the invention but are intended to be exemplary only. The reagents such as kit and buffer used in the method are only specifically selected reagents in the specific embodiment, and it should be understood that other corresponding reagents can be selected by those skilled in the art according to the needs to achieve the purpose of the invention.
Example 1: a preferred embodiment of the composition of the kit of the invention
The genetic sequences of Bordetella pertussis nucleic acids and drug-resistant mutation sites in Genbank and domestic and foreign documents are analyzed by using Blast tool, a gene promoter region specific to Bordetella pertussis toxin and a single-base mutation site of a 23S rRNA variable region A2047G of a Bordetella pertussis drug-resistant region are respectively selected as detection target sequences, a plurality of sets of primers and probes are designed and synthesized aiming at the two detection target sequences, and specific primers and probes are designed aiming at a conserved region of a positive quality control product human beta-globin gene, and the result is shown in Table 1. The primers and the probes are synthesized by biological engineering (Shanghai) GmbH, and comprise detection probes of bordetella pertussis (the 5 'end is marked with FAM fluorescent reporter group, and the 3' end is marked with BHQ1 fluorescent quenching group); a bordetella pertussis drug-resistant mutation site probe (5 'end is marked with VIC fluorescent reporter group, 3' end is marked with BHQ1 fluorescent quenching group); an internal standard beta-globin gene (HBB) probe, (a fluorescence reporter group is CY5, and a fluorescence quenching group is BHQ 2).
The kit for simultaneously detecting bordetella pertussis and the drug-resistant mutation site thereof comprises a fluorescent PCR reaction solution and an internal standard for detecting bordetella pertussis.
The fluorescent PCR reaction solution comprises the following components:
(1) a primer group: the concentration of each primer in the primer group is 0.6 mu M; the primer group comprises: primer BP-F4: a forward primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.1 in a sequence table; primer BP-R4: a reverse primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.2 in a sequence table; primer MBP-F4: a forward primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.4 in a sequence table; primer MBP-R2: a reverse primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.5 in a sequence table;
(2) and (3) probe group: the concentration of each probe in the probe group is 0.3 mu M; the probe set comprises: probe BP-P2-FAM: a probe for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.3 in a sequence table; probe MBP-P2-VIC: a probe for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.6 in a sequence table;
(3) taq DNA polymerase: the concentration is 1.5U/reaction;
(4) reaction buffer: dNTP concentration 0.2mM, MgCl2The concentration is 2.5mM, the final concentration of KCl is 390mM, the pH of Tris-HCl is 9.39, and the final concentration is 250 mM;
(5) primer probes of the internal standard: the forward primer beta-Globin-F1 is shown as SEQ ID NO.7 in the sequence table; the reverse primer beta-Globin-R2 is shown as SEQ ID NO.8 in the sequence table; the probe beta-Globin-CY 5 is shown as SEQ ID NO.9 in the sequence table.
The internal standard is a beta-globin gene (HBB) fragment.
TABLE 1 primer and Probe sequence Listing
The names of primers and probes in Table 1 are explained below:
BP-F4: a forward primer for bordetella pertussis nucleic acid;
BP-R4: a reverse primer for bordetella pertussis nucleic acid;
MBP-F4: detecting a positive primer of a bordetella pertussis drug-resistant mutation site;
MBP-R2: detecting a reverse primer for a bordetella pertussis drug-resistant mutation site;
beta-Globin-F1: a β -globin gene (HBB) forward primer;
beta-Globin-R2: beta-globin gene (HBB) reverse primer;
BP-P2-FAM: a pertussis nucleic acid detection probe sequence;
MBP-P2-VIC: detecting a probe sequence of pertussis drug-resistant mutation points;
beta-Globin-CY 5: detection probe sequence of beta-globin gene (HBB).
Example 2: second preferred embodiment of the kit of the invention
Based on example 1, the bordetella pertussis nucleic acid and the detection kit for drug-resistant mutation sites (Taqman fluorescence probe method) of this example have the following composition:
the kit can also be matched with a quantitative standard kit according to requirements.
Example 3: the invention establishes a pertussis nucleic acid and a PCR detection method of drug-resistant mutation sites
The invention establishes a PCR detection method of bordetella pertussis nucleic acid and drug-resistant mutation sites, and the preferred embodiment is as follows:
1. extraction of genome DNA of sample to be detected
Taking 50 mu L of oropharyngeal swab to-be-detected sample and negative control (Hep-2), centrifuging for a short time, taking supernatant, extracting nucleic acid by using nucleic acid extraction or purification reagent (Yuexiu mechanical preparation No. 20150194) produced by Guangdong and Xin health science and technology limited company, and strictly operating according to the instruction.
2. 20 mu L of bordetella pertussis and preparation of drug resistance detection amplification reaction liquid
20 mu L of bordetella pertussis and amplification reaction solution for drug resistance detection were prepared as follows:
(1) primer set in example 1 and primers of internal standard: the concentration is 0.6 mu M;
(2) probe set in example 1 and probe of internal standard: the concentration is 0.3 mu M;
(3) taq DNA polymerase: the concentration is 1.5U/reaction;
(4) reaction buffer: dNTP concentration 0.2mM, MgCl2The concentration was 2.5mM, the final concentration of KCl was 390mM, and the final concentration of Tris-HCl (pH 9.39) was 250 mM.
3. Real-time fluorescent quantitative PCR detection
And (3) adding 5 mu L of the sample DNA to be detected obtained in the step (1) into the reaction solution prepared in the step (2), tightly covering a tube cover, and centrifuging for a short time. And (3) placing the PCR reaction tube into a fluorescent PCR amplification instrument for amplification detection. And simultaneously detecting signal values of three types of fluorescence, namely FAM/VIC/CY5, wherein FAM fluorescence indicates Bordetella pertussis, VIC fluorescence indicates drug-resistant mutation (A2047G), CY5 fluorescence indicates positive quality control substance beta-globin, and fluorescence collection is carried out in the process of reacting at 55 ℃ for 35 seconds in each cycle. The specific cycle parameters are set as follows:
4. analysis of the reaction results according to the software of the relevant instruments
(1) Setting analysis conditions: the method comprises the steps of adjusting a start value and an end value of a Baseline (Baseline) according to an analyzed image, automatically or manually adjusting a threshold value according to a test condition, wherein the variable range of the start value is 1-10 and the variable range of the end value is 5-20 under a general condition.
(2) The recording instrument automatically analyzes the calculated Ct value of the sample.
(3) And (4) interpretation of results:
and I, detection is invalid: if the internal standard is detected to be negative, namely the Ct value of the internal standard is more than 36.00, the detection of the sample is invalid, the reason should be searched and eliminated, and the sample is subjected to repeated tests.
And II, detection is effective: the positive control obviously increases fluorescence signals of fluorescence channels of FAM and VIC, presents a typical S-shaped curve, and Ct values are both less than or equal to 30.00; there was no significant increase in fluorescence signal at CY5 and no significant sigmoidal amplification curve.
When the Ct value of the fluorescence signal FAM channel of the bordetella pertussis is more than 26.00, the result is judged according to the following steps:
when the internal standard is detected to be positive, namely the Ct value is less than or equal to 36.00, the fluorescence signal of the FAM fluorescence channel is obviously increased and is in a typical S-shaped curve, the Ct value is less than or equal to 36.00, and the VIC has no obvious amplification curve; the result shows that the sample to be detected is Bordetella pertussis DNA, but the A2047G drug resistance mutation does not occur.
When the internal standard is detected to be positive, namely the Ct value is less than or equal to 36.00, the fluorescence signals of the FAM and VIC fluorescence channels are obviously increased and present a typical S-shaped curve, and the Ct value is less than or equal to 36.00; the result shows that the sample to be detected is Bordetella pertussis DNA and the A2047G drug resistance mutation occurs.
When the internal standard detection is positive, namely the Ct value is less than or equal to 36.00, the Ct value of the FAM fluorescence channel is more than 36.00 and no typical S-shaped curve exists, and the fact that the DNA of the bordetella pertussis is not contained in the sample to be detected is shown.
And IV, when the Ct value of the FAM channel is less than or equal to 26.0, carrying out result interpretation according to the following steps:
when the internal standard detection is positive, namely the Ct value is less than or equal to 36.00, the fluorescence signal of the FAM fluorescence channel is obviously increased and presents a typical S-shaped curve, and when the Ct value of the FAM channel is less than or equal to 26.00, a weaker amplification curve is presented in the VIC fluorescence channel, whether the bordetella pertussis is mutated and positive or not needs to be distinguished by calculating the Ct difference value delta Ct of the FAM and the VIC:
when the delta Ct is less than or equal to 6, the DNA of the sample to be detected is Bordetella pertussis and A2047G drug-resistant mutation occurs;
and when the delta Ct is more than 6, the DNA of the sample to be detected is Bordetella pertussis and the A2047G drug resistance mutation does not occur.
If quantitative detection is needed, a standard curve can be drawn according to the Ct values of 5 quantitative standard products with concentration gradients and 5 concentrations, and the Ct value of the sample to be detected is substituted into the standard curve to obtain a quantitative detection result.
Example 4: the invention discloses a Bordetella pertussis nucleic acid, a method for detecting drug-resistant mutation sites and application of a kit
1. Specificity verification
After inactivation of Bordetella parapertussis, Bordetella bronchiseptica, and clinically isolated Streptococcus pneumoniae, Haemophilus influenzae, adenovirus type 3, adenovirus type 7, cytomegalovirus, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, rhinovirus, parainfluenza virus type 1, parainfluenza virus type 3, respiratory syncytial virus type A, Mycoplasma pneumoniae, Lactobacillus casei, Escherichia coli, Proteus, Shigella flexneri purchased from ATCC according to the method of example 3, 50uL of the pathogen solution to be detected and the negative and positive controls of the kit are taken, nucleic acid is extracted by a nucleic acid extraction or purification reagent (Yueju mechanical equipment No. 20150194) produced by Guangdong and Xinsheng health technology Limited, and the kit described in example 1 is used for detection to verify whether the kit can specifically detect only Bordetella pertussis.
The results are shown in fig. 1, fig. 1 is a graph showing the amplification curve of positive control bordetella pertussis and other pathogens in the specificity test, and as can be seen from fig. 1, the amplification curve of the cross reaction test is not sterile except for the positive control bordetella pertussis. The results in FIG. 1 show that other pathogens having the same or similar infection symptoms as those of the same genus and infection sites, including Bordetella parapertussis, Bordetella bronchiseptica, Streptococcus pneumoniae, Haemophilus influenzae, adenovirus type 3, adenovirus type 7, cytomegalovirus, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, rhinovirus, parainfluenza virus type 1, parainfluenza virus type 3, respiratory syncytial virus type A, Mycoplasma pneumoniae, Lactobacillus casei, Escherichia coli, Proteus, Shigella flexneri, do not cross-react with each other in the specificity of the detection kit.
2. Sensitivity detection
To a concentration of 105The reference substance of CFU/mu L Bordetella pertussis enterprises is 105、104、103、10220, 10 CFU/mu L gradient dilution is used as a sample to be detected; to a concentration of 103The reference substance of CFU/mu L drug-resistant Bordetella pertussis enterprises is 103、10210 CFU/mu L gradient dilution is used as a sample to be detected; detection was carried out according to the method of example 3.
Results are shown in fig. 2 and 3, and fig. 2 is a result of a sensitivity test for bordetella pertussis at different gradient concentrations; FIG. 3 shows the results of sensitivity tests for drug-resistant B.pertussis in different gradient concentrations; as can be seen from the results of FIGS. 2 and 3, the lowest detection limit of the method is about 10 CFU/. mu.L, and the method has higher sensitivity.
3. Precision (repeatability) test
To a concentration of 104CFU/mu L Bordetella pertussis enterprise reference and 103The precision detection test is carried out for 20 times by taking CFU/mu L drug-resistant Bordetella pertussis enterprise reference product gradient dilution as a sample to be tested, and the specific test steps are as follows:
(1) extraction 104CFU/mu L Bordetella pertussis enterprise reference and 103CFU/mu L of drug-resistant Bordetella pertussis enterprise reference nucleic acid;
(2) samples at this concentration level were tested in 20 replicates using the method described in example 3.
(3) And (4) analyzing results: the measurement result was subjected to Coefficient of Variation (CV) calculation, CV (%). SD/MN 100%, (SD is the standard deviation; MN is the average value)
The results are shown in FIGS. 4 and 5, and 10 in FIG. 44Results of precision test of bordetella pertussis at CFU/μ L concentration; FIG. 5 is 103Results of precision test of CFU/. mu.l concentration of drug-resistant bordetella pertussis; as can be seen from FIG. 4, the Ct values of 20 detection results are all between 21.85 and 24.15; as can be seen from FIG. 5, the Ct values of 20 detection results are all between 24.7 and 27.3; the coefficient of variation CV (%) of the detection results is less than 5%, and the precision is good.
4. Simulating the detection of clinical samples and determining the positive judgment value of delta Ct
The inventor finds that the Ct value of the VIC channel of wild-type Bordetella pertussis is larger than that of the FAM channel at high concentration, namely the difference between Ct values of the two fluorescence channels is defined as delta Ct ═ VIC-FAM.
The concentration is 1.0X 106The CFU/μ L wild-type bordetella pertussis bacterial solution was diluted with a clinical negative sample and used as a sample to be tested, and three batches of the kit shown in example 2 were used for the detection, and each batch of the kit repeated 62 times the detection on the sample, and table 2 shows the detection results of one batch of the kit:
TABLE 2 determination of the Positive judgement of the Delta Ct values Using the first batch of kits to simulate clinical samples
ROC curve analysis was performed on 62 data in this example using SPSS software to determine a positive determination of Δ Ct. The above statistical results of ROC curve analysis show that the Ct difference (delta Ct) corresponding to the maximum Youden index is 6.005, and the whole is 6.
The detection result of the sample to be detected after repeated detection for 62 times shows that when the Ct of the FAM channel is less than or equal to 26.0, the result that the Ct difference value of the two channels is more than 6.0 is met, and accordingly, when the wild Bordetella pertussis is high in concentration (the Ct is less than or equal to 26.0) and the Ct difference value delta Ct of the FAM and the VIC is more than 6, the sample can be judged to be positive and not to have mutation, and the misjudgment of the sample as drug resistance is avoided.
5. Optimization experiment of primer probes
1) Designing a control primer and a control probe;
2) comparative primer probes designed according to different positions of a promoter target region of a bordetella pertussis gene are as follows:
the sequence of the upstream primer is as follows: P-F1: 5'-GCATGAACGCTCCTTCG-3', respectively;
the sequence of the downstream primer is as follows: P-R1: 5'-ATCCCGTCTTCCCCTCTG-3', respectively;
the probe sequence is as follows: P-P1: FAM-5'-CGTGCTGACCCCCCTGCCA-3' BHQ 1.
25 clinical samples of Bordetella pertussis (sequencing result positive for Bordetella pertussis) were tested according to the method of example 2, and the results of parallel experiments using the primer pair for detecting Bordetella pertussis and the probe (BP-F4, BP-R4, BP-P2-FAM) of example 2 are shown in FIG. 6, and FIG. 6 is a graph showing the optimized experiment results of the primer probe in the present test method. As can be seen from fig. 6, only 15 of the 25 bordetella pertussis clinical samples were positive, indicating that the positive detection rate of the comparative primer probe was much lower than that of example 2 of the present invention. Furthermore, when the detection was carried out in accordance with the method of example 2 while carrying out the parallel experiments using the primer set for detecting B.pertussis and the probe (BP-F4, BP-R4, BP-P2-FAM) of example 2, the detection sensitivity of the comparative primer probe was 100 CFU/. mu.L, which was also lower than the sensitivity of 10 CFU/. mu.L of example 2 of the present invention (see the 2 nd experiment: sensitivity detection experiment in this example). The above shows that the primer probe optimized in example 2 is the best choice for the sensitivity of the present invention.
Although the preferred embodiments of the present invention have been described, it will be understood by those skilled in the art that the present invention is not limited to the embodiments disclosed, and all changes and modifications that come within the spirit of the invention are desired to be protected. For example, other labeling systems than Taqman technology are used, such as fluorescent labeling technologies like molecular beacon MB probe, MGB probe, fluorescent double-hybrid probe, etc.; or other labeling substances than the fluorescence group and the fluorescence quenching group, such as ROX, TET, HEX, JOE, TEXAS RED and the like, and a high resolution melting curve method using dye mosaic SYBR Green and other saturated dsDNA dyes, and furthermore, the use of the specific primer probe sequence described in the present invention, or the homology of the primer probe related to the present invention exceeding 75%, are within the scope of the present invention, because the changes and the replacement of the conventional means known to those skilled in the art are within the scope of the present invention.
Sequence listing
<110> Guangdong and Xin health science and technology Limited
<120> method and kit for simultaneously detecting bordetella pertussis and drug-resistant mutation sites thereof
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ggtgcctatt ttacggatca ca 22
<210> 1
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<212> BP
<213> BP-P2-FAM
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tcccgctact gcaatccaac acg 23
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<213> β-Globin-R2
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cacagggtga ggtgtaagtg at 22
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<213> β-Globin-CY5
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Claims (9)
1. A method for simultaneously detecting Bordetella pertussis and drug-resistant mutation sites thereof comprises the following steps: adding the DNA and the internal standard of a sample to be detected into a fluorescent PCR reaction solution for amplification reaction, and then judging the reaction result according to the Ct value of a fluorescent signal detection channel, wherein the judgment is carried out according to the following method:
(1) when the Ct value of the bordetella pertussis nucleic acid fluorescence signal detection channel is more than 26.00, the identification is carried out according to the following steps:
when a Ct value detected by an internal standard is less than or equal to 36.00, a fluorescence signal of a fluorescence signal detection channel of bordetella pertussis nucleic acid increases and is in a typical S-shaped curve, the Ct value is less than or equal to 36.00, and a fluorescence signal of the fluorescence signal detection channel of a bordetella pertussis drug-resistant mutation site has no obvious amplification curve, the DNA of the sample to be detected is bordetella pertussis DNA, but the A204 2047G drug-resistant mutation does not occur;
when the Ct value detected by an internal standard is less than or equal to 36.00, the fluorescence signals of the detection channels of the bordetella pertussis nucleic acid and the bordetella pertussis drug-resistant mutation site fluorescence signals are increased and are in typical S-shaped curves, and the Ct values are less than or equal to 36.00, the DNA of the sample to be detected is bordetella pertussis DNA, and A2047G drug-resistant mutation occurs;
when the Ct value detected by the internal standard is less than or equal to 36.00, the Ct value of a detection channel of the bordetella pertussis nucleic acid fluorescence signal is greater than 36.00 and no typical S-shaped curve exists, the DNA of the sample to be detected is not the bordetella pertussis DNA;
(2) when the Ct value of the bordetella pertussis nucleic acid fluorescence signal detection channel is less than or equal to 26.00, judging according to the following steps:
when the Ct value of an internal standard detection channel is less than or equal to 36.00, the fluorescence signal of a Bordetella pertussis nucleic acid fluorescence signal detection channel is increased and is in a typical S-shaped curve, and when the Ct value of the Bordetella pertussis nucleic acid fluorescence signal detection channel is less than or equal to 26.00, the Bordetella pertussis drug-resistant mutation site fluorescence signal detection channel has an amplification curve; then, whether the bordetella pertussis is mutated or not needs to be distinguished by calculating the Ct difference delta Ct between the bordetella pertussis nucleic acid fluorescence signal and the bordetella pertussis drug-resistant mutation site fluorescence signal:
when the delta Ct is less than or equal to 6, the DNA of the sample to be detected is Bordetella pertussis and A2047G drug-resistant mutation occurs;
and when the delta Ct is more than 6, the DNA of the sample to be detected is Bordetella pertussis and the A2047G drug resistance mutation does not occur.
2. The method of claim 1, wherein the fluorescent PCR reaction solution comprises the following components:
(1) a primer group: the concentration of each primer in the primer group is 0.6 mu M;
(2) and (3) probe group: the concentration of each probe in the probe group is 0.3 mu M;
(3) taq DNA polymerase: the concentration is 1.5U/reaction;
(4) reaction buffer: dNTP concentration 0.2mM, MgCl2The concentration was 2.5mM, the final concentration of KCl was 390mM, the pH of Tris-HCl was 9.39, and the final concentration was 250 mM.
3. The method of claim 2, wherein the primer set comprises:
primer BP-F4: a forward primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.1 in a sequence table;
primer BP-R4: a reverse primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.2 in a sequence table;
primer MBP-F4: a forward primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.4 in a sequence table;
primer MBP-R2: the reverse primer for detecting the bordetella pertussis drug-resistant mutation site is shown as a sequence in SEQ ID NO.5 in a sequence table.
4. The method of claim 2, wherein the set of probes comprises:
probe BP-P2-FAM: a probe for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.3 in a sequence table;
probe MBP-P2-VIC: a probe for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.6 in a sequence table;
the bordetella pertussis nucleic acid fluorescence signal detection channel is a fluorescence detection channel of FAM in the probe BP-P2-FAM;
the Bordetella pertussis drug-resistant mutation site fluorescence signal detection channel is a VIC fluorescence detection channel in the probe MBP-P2-VIC.
5. The method according to claim 1, wherein the internal standard is β -globin gene (HBB) and the primer probes of the internal standard are:
the forward primer beta-Globin-F1 is shown as SEQ ID NO.7 in the sequence table;
the reverse primer beta-Globin-R2 is shown as SEQ ID NO.8 in the sequence table;
the probe beta-Globin-CY 5 is shown as SEQ ID NO.9 in the sequence table.
6. The method of claim 1, wherein the amplification reaction conditions are:
94℃，2min；
entering a circulation stage: denaturation at 94 ℃ for 10s, annealing and extension at 55 ℃ for 35s, and reaction for 40 cycles.
7. Kit for use in the method for simultaneous detection of bordetella pertussis and its resistant mutation sites according to one of claims 1 to 6, characterized in that it comprises a fluorescent PCR reaction solution for detection of bordetella pertussis and an internal standard.
8. The kit according to claim 7, wherein the fluorescent PCR reaction solution comprises the following components:
(1) a primer group: the concentration of each primer in the primer group is 0.6 mu M; the primer group comprises:
primer BP-F4: a forward primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.1 in a sequence table;
primer BP-R4: a reverse primer for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.2 in a sequence table;
primer MBP-F4: a forward primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.4 in a sequence table;
primer MBP-R2: a reverse primer for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.5 in a sequence table;
(2) and (3) probe group: the concentration of each probe in the probe group is 0.3 mu M; the probe set comprises:
probe BP-P2-FAM: a probe for detecting nucleic acid of bordetella pertussis, which has a sequence shown as SEQ ID NO.3 in a sequence table;
probe MBP-P2-VIC: a probe for detecting a bordetella pertussis drug-resistant mutation site, which has a sequence shown as SEQ ID NO.6 in a sequence table;
(3) taq DNA polymerase: the concentration is 1.5U/reaction;
(4) reaction buffer: dNTP concentration 0.2mM, MgCl2The concentration is 2.5mM, the final concentration of KCl is 390mM, the pH of Tris-HCl is 9.39, and the final concentration is 250 mM;
(5) primer probes of the internal standard:
the forward primer beta-Globin-F1 is shown as SEQ ID NO.7 in the sequence table;
the reverse primer beta-Globin-R2 is shown as SEQ ID NO.8 in the sequence table;
the probe beta-Globin-CY 5 is shown as SEQ ID NO.9 in the sequence table.
9. The kit according to claim 7, characterized in that the internal standard is a fragment of the β -globin gene HBB.
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