CN113186308B - RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting drug resistance of brucella amikacin and using method thereof - Google Patents
RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting drug resistance of brucella amikacin and using method thereof Download PDFInfo
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Abstract
An RT-PCR kit for detecting brucella amikacin resistance and a using method thereof, belonging to the technical field of biology. The kit comprises a solution A (RT-PCR buffer solution, RT-DNA polymerase and sterilized double distilled water), a solution B (primer pair, probe), a negative quality control standard substance and a positive quality control standard substance, wherein the kit comprises specific primer pairs seq ID No.1 and seq ID No.2 and probe seq ID No.3 for detecting brucella, and specific primer pairs seq ID No.4 and seq ID No.5 and probe seq ID No.6 for detecting drug resistance of brucella amikacin. The RT-PCR kit has strong practicability, short time consumption and good repeatability, and can quickly, effectively and visually identify whether clinical samples contain brucella with amikacin resistance after RT-PCR amplification.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an RT-PCR (reverse transcription-polymerase chain reaction) kit for detecting drug resistance of brucella amikacin and a using method thereof
Background
Brucella is a facultative intracellular parasitic bacterium belonging to the group B bioterrorism agent, causes persistent infection of a host by the immune system parasitic to the host, and can cause a global zoonosis-brucellosis (hereinafter referred to as brucellosis). The public health and safety are affected by the cloth diseases, which are mainly manifested as fever, hyperhidrosis, debilitation, arthralgia, etc., serious patients lose the labor capacity of patients, and finally the economic development is affected [1 ]. The "method for preventing and treating infectious diseases of the people's republic of China" classifies brucellosis, SARS, anthrax, AIDS, rabies, etc. as infectious diseases of type B. Brucella, which is a common cause of infection with human brucella, is mainly of the sheep (b.melitensis), cattle (b.abortus), pig (b.suis) and dog (b.canis) species. According to statistics, the incidence rate of the global brucellosis is more than 50 ten thousand in average year, the incidence rate of the brucellosis in each million population in some epidemic countries is more than 100[2], the incidence rate of the brucellosis in each million population in the middle east is more than 200, and the highest syrian incidence rate (1603.4), but according to the survey of the World Health Organization (WHO), the actual incidence rate is 10-25 times of the report [3 ]. The brucellosis in China is firstly reported in inner Mongolia [4], and the epidemic situation of the brucellosis between people occurs twice epidemic peaks in 1957-1963 and 1969-1971. Since the mid 90 s of the 20 th century, brucellosis has been abused again domestically and expanded from north to south [5] disease cases have been reported in 32 municipalities and municipalities throughout the country [6 ]. The existing research shows that the brucellosis in China is mainly distributed in provinces and autonomous areas with developed animal husbandry such as inner Mongolia and Xinjiang, and the infection and incidence rate of farmers, herders, veterinarians, slaughter workers and the like are high.
The brucella drug sensitivity detection needs to be carried out in a biosafety three-level laboratory, and for a long time, little work is done in the brucella drug resistance detection aspect in China. Current research indicates that some rifampicin-resistant Brucella strains appear worldwide [7 ]. Amikacin is used as a medicine for treating brucellosis in partial areas, and if the brucella infected by patients has amikacin resistance, the amikacin seriously affects the brucellosis treatment of the patients. The method utilizes the correlation between the drug-resistant phenotype and the genotype of the brucella abortus isolate amikacin, analyzes the drug-resistant mechanism of the amikacin at the genome level, combines an RT-PCR method, screens a truly feasible drug-resistant gene detection sequence, and is used for detecting the resistance of the brucella amikacin. The invention relates to common instruments, has low cost, can determine the amikacin resistance of the strain by visual inspection of a fluorescence amplification curve without sequencing, can be applied to clinical identification, and practically provides technical support for the drug sensitivity level of brucella in China.
Reference to the literature
1.Nelson-Jones A.Brucellosis[J].Postgrad Med J,1952.28(324):529-34.https://doi.org/10.1136/pgmj.28.324.529
2.Bukhari E E.Pediatric brucellosis.An update review for the new millennium[J].Saudi Med J,2018.39(4):336-341.https://doi.org/10.15537/ smj.2018.4.21896
3.Hasanjani Roushan M R and Ebrahimpour S.Human brucellosis:An overview[J].Caspian J Intern Med,2015.6(1):46-7.
4.Zia S H and Wang F L.Brucellosis in north China；a clinical,etiological and epidemiological study[J].Am J Trop Med Hyg,1949.29(6):925-36.https://doi.org/10.4269/ajtmh.1949.s1-29.925
5.Piao D R,Liu X,Di D D,et al.Genetic polymorphisms identify in species/biovars of Brucella isolated in China between 1953and 2013by MLST[J].BMC Microbiol,2018.18(1):7.https://doi.org/10.1186/s12866-018-1149-0
6.Li Y J,Li X L,Liang S,et al.Epidemiological features and risk factors associated with the spatial and temporal distribution of human brucellosis in China[J].BMC Infect Dis,2013.13:547.https://doi.org/10.1186/ 1471-2334-13-547
7.Barbosa Pauletti R,Reinato Stynen A P,Pinto da Silva Mol J,et al.Reduced Susceptibility to Rifampicin and Resistance to Multiple Antimicrobial Agents among Brucella abortus Isolates from Cattle in Brazil[J].PLoS One,2015.10(7):e0132532.https://doi.org/10.1371/ journal.pone.0132532。
Disclosure of Invention
The inventor finds that certain isolated strain has amikacin resistance through the detection of common brucellosis therapeutic drug resistance tests. The inventors obtained the whole genome sequence of the isolate by whole genome sequencing. Through whole genome sequence comparison, an amikacin resistance gene is obtained, a specific primer pair seq ID No.4 and seq ID No.5 and a specific probe seq ID No.6 aiming at the gene are designed according to the invention, brucella identification primers seq ID No.1 and seq ID No.2 and a probe seq ID No.3 are combined, and brucella amikacin resistance strains can be identified by combining the amplification results of the two groups of probes. The above is the technical basis for the present invention.
The invention aims to provide an amikacin resistant strain capable of quickly and effectively identifying brucellosis in clinical samples, which is used for solving the problem of quickly identifying the amikacin resistant strain in the clinical and prevention and control processes of brucellosis. The invention relates to common instruments, can determine whether the infection is the brucella resistant to the amikacin by visual inspection without sequencing, and can be applied to clinical identification.
In order to realize the purpose of the invention, the following technical scheme is adopted:
the PCR kit consists of a solution A, a solution B, a positive quality control standard substance and a negative quality control standard substance; the solution A comprises RT-PCR buffer solution, dNTP solution with the concentration of 2.5mM and Mg with the concentration of 5mM2+The solution, 2.5U/. mu.l RT-DNA polymerase and sterilized double distilled water, wherein each 750. mu.L solution A correspondingly comprises RT-PCR buffer solution 75. mu. L, dNTP solution 75. mu. L, Mg2+Solution 75. mu. L, RT-DNA polymerase 25. mu.L, sterilized double distilled water 500. mu.L; the solution B comprises specific primer pairs seq ID No.1 and seq ID No.2 and a probe seq ID No.3 for detecting brucella, and specific primer pairs seq ID No.4 and seq ID No.5 and a probe seq ID No.6 for detecting drug resistance of brucella amikacin, the concentration of each primer and each probe in the solution B is 0.2 mu M, the solvent is water, and each primer and each probe are respectively:
seq ID No.1：5’-AATGCGATCAAGTCGGGCG-3’
seq ID No.2：5’-TGCCATCATAAAGGCCGGTG-3’
seq ID No.3：5’FAM-TGCCATCATAAAGGCCGGTG-3’BHQ-1
seq ID No.4：5’-AACTGTTCGCCAGGCTCAAG-3’
seq ID No.5：5’-AAGCGGCCATTTTCCACCAT-3’
seq ID No.6：5’VIC-TCGTGACCCATGGCGATGCCTGCTT-3’BHQ-2
the positive quality control standard substance is adjusted in specific turbidity by drug-resistant strain (amikacin resistant brucella strain), and the bacterium content is about 1.5 × 105CFU/mL, and inactivating at 80 deg.C for 60 min;
the negative quality control standard substance is prepared from sterilized double distilled water.
An RT-PCR kit for detecting brucella amikacin resistance and a using method thereof comprise the following steps:
(1) extracting the genome DNA of the sample to be detected, and detecting the concentration by a nucleic acid concentration detector Nanodrop to be higher than 1 ng/muL. The sample to be tested includes but is not limited to blood, serum, milk sample, tissue sample, aerosol sample. The corresponding commercial kit or extraction reagent can be selected according to the sample to be detected.
(2) Mixing the solution A and the solution B in proportion by taking the extracted sample genome DNA as a template, adding the template, simultaneously carrying out control on a positive quality control standard substance and a negative quality control standard substance, and carrying out fluorescent quantitative detection; the fluorescent quantitative detector takes a LightCycler 480 instrument as an example; at least three test samples: one solution A + solution B + DNA template to be tested and extracted, one solution A + solution B + positive quality control standard, and one solution A + solution B + negative quality control standard;
the PCR amplification system of the three test samples is 25 mu L/sample, and specifically comprises the following steps: 15 mu L A liquid, 7 mu L B liquid, and 3 mu L of DNA template to be tested and extracted or positive quality control standard or negative quality control standard;
the RT-PCR is carried out by a two-step method, and the fluorescent quantitative amplification program comprises the following steps: 30s at 95 ℃, 5s at 95 ℃ and 30s at 60 ℃, and 40 cycles;
(3) and (4) judging a result: wherein, both FAM and VIC signals of the sample corresponding to the positive quality control standard substance in the step (2) have amplification curves, the CT value is between 32 and 35, and the FAM and VIC signals of the sample corresponding to the negative quality control standard substance in the step (2) have no amplification curves, so the test is successful; otherwise, the test fails and needs to be redone;
(4) after the test in the step (3) is successful, corresponding FAM and VIC signals of the sample of the DNA template to be tested and extracted: if the FAM signal of the extracted DNA template to be tested has an amplification curve and the VIC signal does not have an amplification curve, the sample to be tested is brucella but does not have amikacin resistance; if the FAM signal has an amplification curve and the VIC signal has an amplification curve, the sample to be detected is brucella and amikacin resistance exists; if the FAM signal has no amplification curve and the VIC signal has an amplification curve, the sample to be detected is non-brucella but has amikacin resistance; and if the FAM signal has no amplification curve and the VIC signal has no amplification curve, the sample to be detected is non-brucella and has no amikacin resistance.
The kit and the use method thereof directly aim at identifying the amikacin resistant Brucella, rather than obtaining the disease diagnosis result.
The invention provides an RT-PCR kit capable of rapidly, effectively and intuitively identifying whether an amikacin resistant Brucella strain exists in a sample and a using method thereof, which are used for solving the problem of the amikacin resistance in the clinical and prevention and control processes of Brucella disease. The kit can be completely popularized and applied, and is beneficial to promoting the diagnosis, drug sensitivity screening and prevention and control of brucellosis.
Drawings
The kit of FIG. 1 detects gradient diluted amikacin resistant Brucella strains. A is the detection result of FAM signal, and the amplification curves are 10 from left to right5-102CFU/. mu.L; b is the result of VIC signal detection, and the amplification curves are 10 from left to right5-102CFU/μL。
FIG. 2 the kit detects an amikacin-sensitive Brucella strain. A is the detection result of the FAM probe, and the amplification curves from left to right are an amikacin resistant strain sample, an amikacin sensitive strain sample, an amikacin resistant strain diluted sample and a positive quality control standard product respectively; b is a VIC signal detection result, amplification curves from left to right are an amikacin resistant strain sample, an amikacin resistant strain diluted sample and a positive quality control standard, and the VIC signal of the amikacin sensitive strain sample has no amplification curve.
Detailed Description
The following examples are intended to further illustrate the invention but are not intended to limit the scope of the invention.
The following examples were conducted according to conventional test conditions and methods or according to test conditions recommended by the manufacturer.
1. Test materials
The amikacin drug resistance and the amikacin sensitive brucella strains and the like identified by the broth dilution method are recovered, passaged and preserved by infectious disease prevention and control of Chinese disease prevention and control center.
PCR Buffer and dNTP mix (2.5mM) for fluorescent quantitation, and DNA polymerase for probes were purchased from Takara Shuzo (Dalian) Co., Ltd.,
the Genomic DNA Purification Kit genome extraction Kit is purchased from Promega corporation, the PCR primers and probes are synthesized by Biotechnology engineering (Shanghai) GmbH, eight calandria are purchased from Bio-Rad corporation, and other biochemical reagents are imported, subpackaged or domestic analytically pure.
2. Testing instrument
RT-PCR amplification instrument (LoChe company)
Bacteria turbidimeter (merriella).
Example 1
Assembly of RT-PCR detection kit
(1) Preparing RT-PCR reaction solution
a. Designing specific amplification primers and probes, and synthesizing the primers by delivering the primers to the company Limited in the biological engineering (Shanghai), wherein the specific primer sequences are as follows:
seq ID No.1：5’-AATGCGATCAAGTCGGGCG-3’
seq ID No.2：5’-TGCCATCATAAAGGCCGGTG-3’
seq ID No.3：5’FAM-TGCCATCATAAAGGCCGGTG-3’BHQ-1
seq ID No.4：5’-AACTGTTCGCCAGGCTCAAG-3’
seq ID No.5：5’-AAGCGGCCATTTTCCACCAT-3’
seq ID No.6：5’VIC-TCGTGACCCATGGCGATGCCTGCTT-3’BHQ-2
b, preparation of RT-PCR solution:
the solution A comprises RT-PCR buffer solution, dNTP solution and Mg2+The kit comprises a solution RT-DNA polymerase and sterilized double distilled water, wherein the solution B comprises specific primer pairs seq ID No.1 and seq ID No.2 and a probe seq ID No.3 for detecting brucella, and specific primer pairs seq ID No.4 and seq ID No.5 and a probe seq ID No.6 for detecting the drug resistance of brucella amikacin.
(2) Preparing a positive quality control standard:
the positive quality control standard substance is prepared by adjusting the turbidity of the amikacin resistant Brucella to 0.5McF (1.5 multiplied by 10)8CFU/mL) was heat inactivated at 80 ℃ for 60min in a volume of 500. mu.L.
(3) Preparing a negative quality control standard:
the negative quality control standard substance is sterilized double distilled water with the volume of 500 mu L.
(4) PCR amplification and fluorescence detection
The PCR amplification system is 25 mu L/sample, and specifically comprises: 15 mu L A liquid, 7 mu L B liquid, and 3 mu L of positive quality control standard substance/negative quality control standard substance/extracted DNA template.
The fluorescent quantitative amplification procedure is as follows: 30s at 95 ℃, 5s at 95 ℃ and 30s at 60 ℃ for 40 cycles.
(4) And (4) judging a result: the FAM and VIC signals of the positive quality control standard substance have an amplification curve, the CT value is between 32 and 35, the negative quality control standard substance does not have an amplification curve, and otherwise, the test fails and needs to be repeated.
For a sample to be detected, if an amplification curve exists in the FAM signal and an amplification curve does not exist in the VIC signal, the sample to be detected is brucella but amikacin resistance does not exist;
for a sample to be detected, if an amplification curve exists in the FAM signal and an amplification curve exists in the VIC signal, the sample to be detected is brucella and amikacin resistance exists;
for a sample to be detected, an amplification curve does not exist in the FAM signal, and an amplification curve exists in the VIC signal, so that the sample to be detected is not Brucella but has amikacin resistance;
for the sample to be detected, the FAM signal has no amplification curve, and the VIC signal has no amplification curve, so that the sample to be detected is non-brucella and has no amikacin resistance.
Example 2 detection of minimum bacterial content by Brucella Amikacin resistance RT-PCR kit
(1) After 48h of streak culture of the amikacin resistant Brucella strain, the turbidity of the strain is adjusted to 0.5McF (about 1X 10) by a turbidimeter8CFU/mL). Taking 1 mu L of bacterial liquid to carry out 10 times of gradient dilution, and finally obtaining 10 percent of low bacterial content5-101CFU/μL。
(2) And extracting the genome DNA of the sample to be detected by using a genome extraction kit, and detecting the concentration by using a nucleic acid concentration detector to be higher than 1 ng/. mu.L.
(3) And (3) taking the extracted DNA of the sample to be detected as a template, mixing the template, the solution A and the solution B in proportion, simultaneously carrying out negative quality control standard control, and carrying out RT-PCR amplification.
The fluorescent quantitative amplification procedure is as follows: 30s at 95 ℃, 5s at 95 ℃ and 30s at 60 ℃ for 40 cycles.
The PCR amplification system is 25 mu L/sample, and specifically comprises: 15 mu L A liquid, 7 mu L B liquid, and 3 mu L of positive quality control standard substance/negative quality control standard substance/extracted DNA template.
(3) And (4) judging a result: the negative quality control standard has no amplification curve.
As the detection sample is a brucella amikacin resistant strain and a diluted sample, both FAM and VIC signals have amplification curves. From the results in FIG. 1, the content of the detected bacteria was 102In the case of CFU/. mu.L sample, both FAM and VIC signal detection CT values were about 33. The results show that FAM and VIC signals in the kit do not cause problems when detecting the drug-resistant strains of the brucella amikacin, and the minimum content of the detection bacteria is 102CFU/. mu.L. The results also show that FAM and VIC probes have similar amplification efficiencies, and the difference of the amplification CT values is not obvious.
Example 3 kit for detection of unknown Brucella Strain of Amikacin
The RT-PCR kit is adopted to detect the unknown brucella strain of the amikacin, and negative control is set.
The feasibility result of the kit for detecting the unknown brucella of the amikacin is shown in figure 2. The fluorescent amplification curve shows that the negative quality control standard has no amplification curve, the positive quality control standard FAM and VIC signals have amplification curves, and the CT value is 34.16 (the CT value is between 32 and 35), which shows that the test is true. The detection result shows that the sample 1 is an amikacin resistant brucella strain, and both FAM and VIC signals of the sample have amplification curves; the sample 2 is an amikacin resistant diluted sample which is 10 times of the diluted sample of the sample 1 (manual dilution operation); the sample 3 is an amikacin-sensitive Brucella strain, the FAM probe of the strain has an amplification curve, and the VIC probe of the strain does not have an amplification curve.
Sequence listing
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Claims (4)
1. An RT-PCR kit for detecting drug resistance of brucella amikacin is characterized in that the PCR kit consists of solution A, solution B, a positive quality control standard substance and a negative quality control standard substance; the solution A comprises RT-PCR buffer solution, dNTP solution with the concentration of 2.5mM and Mg with the concentration of 5mM2+Solution, 2.5U/. mu.l RT-DNA polymerizationEnzyme and sterile double distilled water, wherein each 750 mu L of solution A correspondingly comprises RT-PCR buffer solution 75 mu L, dNTP solution 75 mu L, Mg2+Solution 75. mu. L, RT-DNA polymerase 25. mu.L, sterilized double distilled water 500. mu.L; the solution B comprises specific primer pairs seq ID No.1 and seq ID No.2 and a probe seq ID No.3 for detecting brucella, and specific primer pairs seq ID No.4 and seq ID No.5 and a probe seq ID No.6 for detecting drug resistance of brucella amikacin, the concentration of each primer and each probe in the solution B is 0.2 mu M, the solvent is water, and each primer and each probe are respectively:
seq ID No.1：5’- AATGCGATCAAGTCGGGCG -3’
seq ID No.2：5’- TGCCATCATAAAGGCCGGTG -3’
seq ID No.3：5’FAM- TGCCATCATAAAGGCCGGTG -3’BHQ-1
seq ID No.4：5’- AACTGTTCGCCAGGCTCAAG -3’
seq ID No.5：5’- AAGCGGCCATTTTCCACCAT -3’
seq ID No.6：5’VIC- TCGTGACCCATGGCGATGCCTGCTT -3’BHQ-2
the positive quality control standard substance is adjusted in specific turbidity by a drug-resistant strain, and the bacterium content is about 1.5 multiplied by 105 CFU/mL, and inactivating for 60min at 80 deg.C;
the negative quality control standard substance is sterilized double distilled water.
2. The use method of the RT-PCR kit for detecting the drug resistance of the Brucella amikacin for the purpose of non-disease diagnosis and treatment according to claim 1, is characterized by comprising the following steps:
(1) extracting genome DNA of a sample to be detected, and detecting the concentration by a nucleic acid concentration detector Nanodrop to be higher than 1 ng/muL; (2) mixing the solution A and the solution B in proportion by taking the extracted sample genome DNA as a template, adding the template, simultaneously carrying out control on a positive quality control standard substance and a negative quality control standard substance, and carrying out fluorescent quantitative detection; the fluorescence quantitative detector takes a LightCycler 480 instrument as an example; at least three test samples: one solution A, solution B and a DNA template to be tested and extracted, one solution A, solution B and a positive quality control standard, and one solution A, solution B and a negative quality control standard;
the PCR amplification system of the three test samples is 25 mu L/sample, and specifically comprises the following steps: 15 mu L A liquid, 7 mu L B liquid, and 3 mu L of DNA template to be tested and extracted or positive quality control standard or negative quality control standard;
the RT-PCR is carried out by a two-step method, and the fluorescent quantitative amplification program comprises the following steps: 30s at 95 ℃, 5s at 95 ℃ and 30s at 60 ℃ for 40 cycles;
(3) and (4) judging a result: wherein, both FAM and VIC signals of the sample corresponding to the positive quality control standard substance in the step (2) have amplification curves, the CT value is between 32 and 35, and the FAM and VIC signals of the sample corresponding to the negative quality control standard substance in the step (2) have no amplification curves, so the test is successful; otherwise, the test fails and needs to be redone;
(4) after the test in the step (3) is successful, corresponding FAM and VIC signals of the sample of the DNA template to be tested and extracted: if the FAM signal of the extracted DNA template to be tested has an amplification curve and the VIC signal does not have an amplification curve, the sample to be tested is brucella but does not have amikacin resistance; if the FAM signal has an amplification curve and the VIC signal has an amplification curve, the sample to be detected is brucella and amikacin resistance exists; and if the FAM signal has no amplification curve and the VIC signal has no amplification curve, the sample to be detected is non-brucella and has no amikacin resistance.
3. The method according to claim 2, wherein the sample to be tested in step (1) is selected from any one of blood, serum, milk sample, tissue sample, and aerosol sample.
4. The method of claim 2, wherein the minimum detectable bacteria content is 102 CFU/μL。
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| CN117947188A (en) * | 2023-12-28 | 2024-04-30 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker, primer, probe kit and application related to brucella streptomycin resistance |
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