CN114369672A - Primer probe composition, kit and method for detecting chlamydia trachomatis - Google Patents
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly provides a primer probe composition, a kit and a method for detecting chlamydia trachomatis. The primer probe composition for detecting the chlamydia trachomatis provided by the invention can be used for quickly amplifying a target gene, and an internal standard is used as an internal control in a PCR amplification system, so that false negative caused by interference substances possibly existing in a sample can be prevented; the kit and the detection method which take the primer probe composition as the main component have the advantages of simple sample treatment process, strong specificity on the chlamydia trachomatis nucleic acid, short time consumption, accurate detection on the chlamydia trachomatis nucleic acid in unknown samples such as genital secretion and the like, no cross reaction with other pathogens which are easy to cause the same or similar clinical symptoms, and the problems of complicated sample treatment, poor specificity and the like in the prior art are solved. The kit has high sensitivity and detection limit of 200copies/ml, and provides reliable experimental basis for diagnosing Chlamydia trachomatis.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a primer probe composition, a kit and a method for detecting chlamydia trachomatis.
Background
Chlamydia trachomatis (Ct) is a prokaryotic microorganism parasitized in living cells, mainly infects epithelial cells of the urinary tract, and is one of the most common pathogens of sexually transmitted diseases at home and abroad in recent years.
The detection for clinically detecting the chlamydia trachomatis infection comprises the following steps: cell culture, smear microscopy, polymerase chain reaction, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, and the like. The isolation culture method is simple to operate and high in specificity, is considered as a gold standard for diagnosing chlamydia trachomatis, but is long in culture time, low in sensitivity and easy to delay the state of an illness; the smear microscopy method is simple and easy to implement, has low cost, but is easily influenced by various factors such as material taking, smear, dyeing and the like, and causes misdiagnosis and missed diagnosis. In recent years, the fluorescence PCR method gradually shows the advantages of the fluorescence PCR method in detection, is a more sensitive, more specific and more accurate nucleic acid detection technology, has accurate detection result and high repeatability, can dynamically reflect the pathogen change before and after treatment of a patient and the clinical relation, and provides more accurate and objective detection result for clinic.
At present, the existing kits for detecting CT-DNA based on real-time fluorescence quantitative PCR technology at home and abroad are applied to clinical detection, the sensitivity is about 500-1000copies/ml, most of the kits lack a perfect quality control system, and the technical level needs to be further perfected and improved, so that the requirements of clinical accurate diagnosis are met.
Disclosure of Invention
The invention aims to solve the problems of low detection sensitivity of the chlamydia trachomatis, lack of a perfect quality control system and the like in the prior art.
Therefore, the invention provides a primer probe composition for detecting chlamydia trachomatis, which comprises a target gene primer probe composition; the target gene primer probe composition comprises:
the sequence of the target gene upstream primer is as follows: GCTCTGCCTGTGGGGAAT, respectively;
the sequence of the target gene downstream primer is as follows: TGCTGATAGCGTCACACCAA, respectively;
the target gene probe has the sequence as follows: TGAACCAAGCCTTATGATCGACGG are provided.
Specifically, the primer probe composition for detecting chlamydia trachomatis further comprises an internal standard primer probe composition; the internal standard primer probe composition comprises:
an internal standard upstream primer with the sequence as follows: TGACAATGTTGGCAAGACTGTTG, respectively;
an internal standard downstream primer with the sequence as follows: GCCGACAAACTGGGAATGAAC, respectively;
an internal standard probe, the sequence of which is: TGGAGATCGTTTTGTGGCTTGTGA are provided.
Specifically, the internal standard is a recombinant plasmid and is used as an internal control in a PCR amplification system to prevent false negative caused by PCR interfering substances possibly existing in a sample, and the sequence of the internal standard is TGACAATGTTGGCAAGACTGTTGATGGAGAGCCTTTTGTGGCTTGTGATTTTTGTTCATTCCCAGTTTGTCGGC.
The invention also provides a kit for detecting chlamydia trachomatis, which comprises a PCR reaction solution; the PCR reaction solution contains the primer probe composition for detecting Chlamydia trachomatis as described in any one of the above.
Specifically, the PCR reaction solution further includes 10 XPCR reaction buffer solution and dNTP.
Specifically, the kit for detecting chlamydia trachomatis further comprises a nucleic acid extracting solution, wherein the nucleic acid extracting solution comprises 10-50mM of Tris, 0.1-1M of NaCl, 0.1-1mM of EDTA, 10-50mM of NaOH, 0.01-0.5mM of sanskatone, 5-20% of Chelex-100 and 0.01-2% of sodium dodecyl sulfate. The nucleic acid extracting solution greatly simplifies the steps of nucleic acid detection and greatly improves the sensitivity.
Specifically, the kit for detecting chlamydia trachomatis further comprises an enzyme mixed solution, wherein the enzyme mixed solution comprises 1-5U/mu l of DNA polymerase and 1-5U/mu l of hot start modified antibody, and can effectively inhibit non-specific annealing of primers and non-specific amplification caused by primer dimers under a low-temperature condition.
Specifically, the kit for detecting chlamydia trachomatis further comprises a positive control and a negative control; the positive control is a strong positive sample of the chlamydia trachomatis; the negative control is an inactivated negative sample without chlamydia trachomatis and herpes simplex virus.
The present invention also provides a method for detecting chlamydia trachomatis, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) carrying out fluorescent quantitative PCR reaction by taking DNA of a sample to be detected as a template, wherein the PCR reaction system comprises any one of the primer probe composition for detecting the chlamydia trachomatis;
(3) after the PCR reaction is finished, the detection result is judged according to the Ct value.
Specifically, the PCR reaction procedure in step (2) is as follows: at 95 ℃ for 3 min; 94 ℃, 15sec, 60 ℃, 30sec, 40 cycles.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the primer probe composition for detecting the chlamydia trachomatis provided by the invention can be used for quickly amplifying a target gene, and the designed internal standard is used as an internal control in a PCR amplification system, so that false negative caused by PCR interfering substances possibly existing in a sample can be prevented. The kit and the detection method for detecting the chlamydia trachomatis nucleic acid, which take the primer probe composition as the main component, have the advantages of simple sample treatment process, strong specificity on the chlamydia trachomatis nucleic acid, less time consumption of the detection method, capability of accurately detecting the chlamydia trachomatis nucleic acid in unknown samples such as genital secretion and the like, no cross reaction with other pathogens which can be sexually transmitted and easily cause the same or similar clinical symptoms, and the problems of complicated sample treatment method, poor kit specificity and the like in the prior art are solved. In addition, the kit has high sensitivity, the detection limit is 200copies/ml, and reliable experimental basis is provided for diagnosing the chlamydia trachomatis.
The present invention will be described in further detail below with reference to the accompanying drawings.
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FIG. 1 shows the results of PCR reaction in example 2 of the present invention.
FIG. 2 shows the results of PCR reaction in example 3 of the present invention; wherein the curves represent 800copies/ml, 400copies/ml, 200copies/ml in order from left to right.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Although representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made thereto without departing from the scope of the invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and equivalents thereof.
The invention provides a primer probe composition for detecting chlamydia trachomatis, which comprises a target gene primer probe composition; the target gene primer probe composition comprises:
the sequence of the target gene upstream primer is as follows: GCTCTGCCTGTGGGGAAT, respectively;
the sequence of the target gene downstream primer is as follows: TGCTGATAGCGTCACACCAA, respectively;
the target gene probe has the sequence as follows: TGAACCAAGCCTTATGATCGACGG are provided.
In order to prevent false negatives caused by PCR interfering substances that may be present in the sample, the primer probe composition further comprises an internal standard primer probe composition; the internal standard primer probe composition comprises:
an internal standard upstream primer with the sequence as follows: TGACAATGTTGGCAAGACTGTTG, respectively;
an internal standard downstream primer with the sequence as follows: GCCGACAAACTGGGAATGAAC, respectively;
an internal standard probe, the sequence of which is: TGGAGATCGTTTTGTGGCTTGTGA are provided.
The sequence of the internal standard is:
TGACAATGTTGGCAAGACTGTTGATGGAGAGCCTTTTGTGGCTTGTGATTTTTGTTCATTCCCAGTTTGTCGGC。
the invention also provides a kit for detecting the chlamydia trachomatis, which comprises PCR reaction liquid, nucleic acid extracting solution, enzyme mixed liquid, positive control and negative control.
Wherein, the PCR reaction solution comprises the primer probe composition for detecting the chlamydia trachomatis, 10 XPCR reaction buffer solution and dNTP.
The nucleic acid extract comprises 10-50mM Tris, 0.1-1M NaCl, 0.1-1mM EDTA, 10-50mM NaOH, 0.01-0.5mM cyperantin, 5% -20% Chelex-100 and 0.01-2% sodium dodecyl sulfate.
The enzyme mixed solution comprises 1-5U/mu l of DNA polymerase and 1-5U/mu l of hot start modified antibody, and can effectively inhibit non-specific annealing of primers and non-specific amplification caused by primer dimer under low temperature condition.
The positive control is a strong positive sample of the chlamydia trachomatis; the negative control is an inactivated negative sample without chlamydia trachomatis and herpes simplex virus.
The present invention also provides a method for detecting chlamydia trachomatis, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) carrying out fluorescent quantitative PCR reaction by taking DNA of a sample to be detected as a template, wherein the PCR reaction system comprises any one of the primer probe composition for detecting the chlamydia trachomatis; the PCR reaction program is: at 95 ℃ for 3 min; 94 ℃, 15sec, 60 ℃, 30sec, 40 cycles.
(3) After the PCR reaction is finished, the detection result is judged according to the Ct value.
The effects of the primer probe composition, the kit and the method for detecting chlamydia trachomatis of the present invention will be examined below with reference to specific examples.
Example 1:
the embodiment provides a kit for detecting chlamydia trachomatis, which comprises the following components:
(1) nucleic acid extracting solution: 25mM Tris, 0.5M NaCl, 0.5mM EDTA, 25mM NaOH, 0.25mM sansantine, 12% Chelex-100, 1% sodium dodecyl sulfonate and 5X 10 concentration5Internal standard template of copies/ml; internal standardA recombinant plasmid of a synthetic 74 base pair DNA sequence for insertion into the pUC18T vector, the sequence being:
TGACAATGTTGGCAAGACTGTTGATGGAGAGCCTTTTGTGGCTTGTGATTTTTGTTCATTCCCAGTTTGTCGGC；
(2) PCR reaction solution: 5 mul 10 XPCR reaction buffer, 0.2mM dNTP, 0.2 muM target gene upstream primer, 0.2 muM target gene downstream primer, 0.2 muM target gene probe, 0.2 muM internal standard upstream primer, 0.2 muM internal standard downstream primer, 0.1 muM internal standard probe; wherein the 10 XPCR reaction buffer comprises 200mM Tris-cl solution at pH 7.5, 30mM magnesium chloride solution, 500mM potassium chloride solution; the dNTPs include dATP, dCTP, dTTP and dGTP;
the sequence of the target gene upstream primer is as follows: GCTCTGCCTGTGGGGAAT, respectively;
the sequence of the target gene downstream primer is as follows: TGCTGATAGCGTCACACCAA, respectively;
the target gene probe sequence is as follows: TGAACCAAGCCTTATGATCGACGG, respectively;
the internal standard upstream primer sequence is as follows: TGACAATGTTGGCAAGACTGTTG, respectively;
the internal standard downstream primer sequence is as follows: GCCGACAAACTGGGAATGAAC, respectively;
the internal standard probe sequence is as follows: TGGAGATCGTTTTGTGGCTTGTGA, respectively;
(3) enzyme mixture liquid: 5U/. mu.l of DNA polymerase and 1U/. mu.l of hot start modified antibody;
(4) a positive control; a strong positive sample of chlamydia trachomatis collected in a clinical hospital;
(5) negative control: does not contain inactivated negative clinical samples of chlamydia trachomatis and herpes simplex virus.
Example 2:
this example provides a method of detecting chlamydia trachomatis using the kit of example 1, comprising the steps of:
(1) adding 35.2 mul of PCR reaction solution and 0.8 mul of enzyme mixed solution into a test tube, mixing uniformly, and centrifuging instantly to obtain a PCR-mix part for later use;
(2) adding 1ml of sterile normal saline into a sample test tube to be tested, fully shaking and shaking up, sucking liquid, transferring the liquid into a 1.5ml centrifuge tube, and centrifuging for 5 minutes at 12000 rpm; discarding the supernatant, adding 50 μ l of nucleic acid extractive solution (because the nucleic acid extractive solution contains solid precipitate particulate matter, the particles are distributed uniformly, and repeatedly sucking with a suction head during each sampling), mixing, centrifuging at 2000rpm for 10sec, water bath at 100 deg.C for 10min, centrifuging at 12000rpm for 10min, and collecting the supernatant of the sample; respectively taking 50 mul of negative control and 50 mul of positive control, adding 50 mul of nucleic acid extracting solution into the negative control and the 50 mul of positive control, uniformly mixing, carrying out water bath at 100 ℃ for 10min, and centrifuging at 12000rpm for 10min for later use;
(3) respectively adding 4 mu l of the supernatant, the negative control and the positive control of the sample to be detected, which are processed in the step (2), into three PCR-mix parts, and putting the test tube into a fluorescence quantitative PCR instrument for PCR amplification, wherein the reaction program is as follows: at 95 ℃ for 3 min; 94 ℃, 15sec, 60 ℃, 30sec, 40 cycles; the detection fluorescence of the target gene is FAM, and the detection fluorescence of the internal standard is HEX/VIC; the results are shown in FIG. 1.
The threshold setting principle is that the threshold line just exceeds the highest point of the normal negative control.
The positive control Ct value is less than 38, the negative control Ct value is countless and the internal standard CT value is less than 40; the fluorescence curve above the threshold should have a pronounced sigmoidal curve, otherwise the experiment should be considered invalid and errors in instrumentation, reagents, amplification conditions, etc. should be checked.
When the method provided by the invention is adopted to detect the chlamydia trachomatis, the judgment standard is as follows:
1. the sample with Ct less than or equal to 38.0 is judged to be positive;
2. the detection sample with Ct more than 38.0 and less than or equal to 40 is recommended to be redone, if the Ct value of the redone result is less than 40, the redone result is positive, otherwise, the redone result is negative;
3. if the CT value measured by the detection sample has no data, the internal standard CT value needs to be checked, the sample with the internal standard CT value less than 40 is a negative sample, and if the internal standard CT value has no data, the false negative condition is suspected, and the test should be repeated.
Example 3:
this example demonstrates the sensitivity of the kit of example 1, as follows:
(1) adding 35.2 mul of PCR reaction solution and 0.8 mul of enzyme mixed solution into a test tube, mixing uniformly, and centrifuging instantly to obtain a PCR-mix part for later use;
(2) diluting the chlamydia trachomatis standard into quantitative standards of 200copies/ml, 400copies/ml and 800copies/ml, and taking the quantitative standards as templates for standby;
respectively taking 50 mul of negative control and 50 mul of positive control, adding 50 mul of nucleic acid extracting solution into the negative control and the 50 mul of positive control, uniformly mixing, carrying out water bath at 100 ℃ for 10min, and centrifuging at 12000rpm for 10min for later use;
(3) and (3) putting 4 mu l of each concentration standard sample, negative control and positive control in the step (2) into a test tube, adding one part of PCR-mix into each test tube, putting the test tubes into a fluorescent quantitative PCR instrument for PCR amplification, wherein the reaction program is as follows: at 95 ℃ for 3 min; 94 ℃, 15sec, 60 ℃, 30sec, 40 cycles; the detected fluorescence of the target gene is FAM, and the detected fluorescence of the internal standard is HEX/VIC.
As shown in the figure 2, when the concentration of the sample is 200copies/ml, the kit provided by the invention can still clearly detect the Chlamydia trachomatis, and the kit has high sensitivity and the detection limit is 200copies/ml, thereby providing reliable experimental basis for diagnosing the Chlamydia trachomatis.
The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.
Claims (10)
1. A primer probe composition for detecting chlamydia trachomatis comprises a target gene primer probe composition; the target gene primer probe composition is characterized by comprising the following components:
the sequence of the target gene upstream primer is as follows: GCTCTGCCTGTGGGGAAT, respectively;
the sequence of the target gene downstream primer is as follows: TGCTGATAGCGTCACACCAA, respectively;
the target gene probe has the sequence as follows: TGAACCAAGCCTTATGATCGACGG are provided.
2. The primer-probe composition for detecting chlamydia trachomatis according to claim 1, further comprising an internal standard primer-probe composition; the internal standard primer probe composition comprises:
an internal standard upstream primer with the sequence as follows: TGACAATGTTGGCAAGACTGTTG, respectively;
an internal standard downstream primer with the sequence as follows: GCCGACAAACTGGGAATGAAC, respectively;
an internal standard probe, the sequence of which is: TGGAGATCGTTTTGTGGCTTGTGA are provided.
3. The primer-probe composition for detecting chlamydia trachomatis according to claim 2, wherein the sequence of the internal standard is:
TGACAATGTTGGCAAGACTGTTGATGGAGAGCCTTTTGTGGCTTGTGATTTTTGTTCATTCCCAGTTTGTCGGC。
4. a kit for detecting chlamydia trachomatis, comprising: comprises a PCR reaction solution; the PCR reaction solution comprises the primer probe composition for detecting Chlamydia trachomatis according to any one of claims 1 to 3.
5. The kit for detecting chlamydia trachomatis according to claim 4, wherein: the PCR reaction solution also comprises 10 XPCR reaction buffer solution and dNTP.
6. The kit for detecting chlamydia trachomatis according to claim 4, wherein: also comprises nucleic acid extracting solution; the nucleic acid extracting solution comprises 10-50mM Tris, 0.1-1M NaCl, 0.1-1mM EDTA, 10-50mM NaOH, 0.01-0.5mM cyperantin, 5% -20% Chelex-100 and 0.01-2% sodium dodecyl sulfate.
7. The kit for detecting chlamydia trachomatis according to claim 4, wherein: also comprises nucleic acid extracting solution; the enzyme mixture comprises 1-5U/. mu.l of DNA polymerase and 1-5U/. mu.l of hot start modified antibody.
8. The Chlamydia trachomatis detection kit of claim 4, wherein: positive and negative controls are also included; the positive control is a strong positive sample of the chlamydia trachomatis; the negative control is an inactivated negative sample without chlamydia trachomatis and herpes simplex virus.
9. A method for detecting chlamydia trachomatis, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) performing a fluorescent quantitative PCR reaction by using DNA of a sample to be detected as a template, wherein the PCR reaction system comprises the primer probe composition for detecting the chlamydia trachomatis according to any one of claims 1 to 3;
(3) after the PCR reaction is finished, the detection result is judged according to the Ct value.
10. The method for detecting chlamydia trachomatis according to claim 9, wherein the PCR reaction procedure in the step (2) is: at 95 ℃ for 3 min; 94 ℃, 15sec, 60 ℃, 30sec, 40 cycles.
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| 陆中奎;: "实时荧光定量PCR检测沙眼衣原体的研究", 热带医学杂志, no. 06, pages 752 - 755 * |
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