CN114921575A - Kit for simultaneously detecting DNA of chlamydia pneumoniae and Chlamydia trachomatis and application thereof - Google Patents

Kit for simultaneously detecting DNA of chlamydia pneumoniae and Chlamydia trachomatis and application thereof Download PDF

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CN114921575A
CN114921575A CN202210737325.2A CN202210737325A CN114921575A CN 114921575 A CN114921575 A CN 114921575A CN 202210737325 A CN202210737325 A CN 202210737325A CN 114921575 A CN114921575 A CN 114921575A
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chlamydia
kit
probe
chlamydia trachomatis
primer
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闫泉
熊永清
张浩淼
甄博文
王莹
梁宁孝
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Aike Ruite Biomedical Technology Nanjing Co ltd
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/295—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Chlamydiales (o)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The invention relates to a kit for simultaneously detecting DNA of chlamydia pneumoniae and Chlamydia trachomatis, wherein the primer group, the probe group and the kit can realize one-time reaction and simultaneously detect the DNA of the chlamydia pneumoniae and the Chlamydia trachomatis, are rare kits for simultaneously detecting two chlamydias at present, have high efficiency and strong specificity and provide a basis for auxiliary diagnosis for clinical examination; through the technical scheme, a new means can be provided for the differential diagnosis of atypical pathogens.

Description

Kit for simultaneously detecting DNA of chlamydia pneumoniae and Chlamydia trachomatis and application thereof
Technical Field
The invention belongs to the field of pathogenic microorganism molecular biology, and particularly relates to a kit for simultaneously detecting DNA of chlamydia pneumoniae and chlamydia trachomatis and application thereof.
Background
Chlamydiae (chlamydia) is a group of very small, non-motile, microorganisms that grow exclusively within cells. Chlamydiae can be divided into 4 kinds, i.e., chlamydia pneumoniae, chlamydia psittaci, chlamydia trachomatis, and chlamydia bovis. Chlamydiae are gram-negative pathogens, a class of prokaryotic microorganisms that pass through bacterial filters, are parasitic in cells, and have a unique developmental cycle. Chlamydia is an organism smaller than bacteria but larger than viruses, is an obligate intracellular parasitic pathogen, similar to bacteria and viruses, with two living loops. It has no ability to synthesize high-energy compounds ATP and GTP, and must be provided by host cells, thus becoming energy parasites, mostly spherical and piled, having cell walls and cell membranes, belonging to prokaryotic cells, and generally parasitizing in animal cells. At present, the chlamydiae are classified into trachoma, pneumonia, parrots, livestock and the like.
The chlamydia pneumoniae is a pathogen which causes atypical pneumonia, can also cause diseases such as bronchitis, pharyngitis, nasosinusitis, otitis media, iritis, myocarditis, endocarditis, meningitis, erythema nodosum and the like, and is also one of important pathogenic bacteria for triggering infection of AIDS and leukemia. In addition, epidemiological and etiological studies suggest that chlamydia pneumoniae infection is also associated with cardiovascular diseases, and the most common is respiratory tract infection caused by chlamydia pneumoniae. The clinical manifestations are more and less typical, generally, pharyngalgia and hoarseness are caused, cough can appear after several days to seven days, low fever is also accompanied, white blood cell count is mostly normal, and blood sedimentation is increased rapidly, the chest radiography can show unilateral segmental pneumonia, even the patients with extensive pathological changes can spread to both lungs and also be accompanied with pleurisy or pleural effusion.
In addition to causing trachoma, chlamydia trachomatis is also a well-recognized source of infection of sexually transmitted diseases. In non-gonococcal urethritis, almost half are caused by chlamydia trachomatis infection. It can also cause urethral syndrome and sexual disease lymphogranuloma, male urethritis, epididymitis, female infertility, cervicitis, pelvic inflammatory disease, etc. Neonatal infection through the birth canal can cause neonatal ophthalmia or neonatal pneumonia. Chlamydia trachomatis can also cause adult pneumonia, is also harmful to pregnant women, and can cause ectopic pregnancy, abortion, dead fetus, chorioamnionitis, premature delivery and the like.
For many years, hospitals and laboratories around the world routinely detect chlamydia by methods such as pathogen isolation and culture, immunological tests, etc., but these detection methods have many defects that are difficult to overcome. The traditional chlamydia separation method has the disadvantages of complicated operation steps, high requirement on conditions, long detection period and relatively difficult guarantee of experimental safety and result accuracy; the immunological detection method usually adopts enzyme-linked immunosorbent assay, has the advantages of intuition, mature technology and good specificity, but has the limitations of time consumption, complex operation, low sensitivity, high false positive and the like on the clinical reference value.
In recent years, the introduction of molecular biology techniques such as real-time fluorescence PCR techniques has greatly improved the current situation of detecting and identifying pathogens. In particular, the multiplex fluorescence quantitative PCR is a multiplex joint inspection developed on the basis of a real-time fluorescence quantitative PCR technology, can realize the amplification and specificity detection of a plurality of gene sequences in the same reaction tube, can simultaneously detect and distinguish chlamydia trachomatis and chlamydia pneumoniae in clinical application, is favorable for accurately judging the state of an illness, and realizes early, quick and accurate treatment of the illness. Successful implementation of multiplex PCR technology requires solving several key technical problems: (1) because multiple primer/probe combinations are required, it is desirable to avoid cross-linking between different primers/probes. (2) Avoiding cross-homology of each set of primers/probes to other target and non-target nucleic acid sequences to prevent false positive results. (3) The optimal amplification conditions for each amplicon are different, and the reaction conditions need to be optimized to ensure that each amplicon can be successfully amplified under a single amplification condition. (4) An internal control is required as a reference to exclude interference from aspects such as nucleic acid extraction.
The PCR method detection product capable of simultaneously detecting and distinguishing the chlamydia trachomatis and the chlamydia pneumoniae in the market at present needs to use 3 pairs of primer probe sets for detection and ensures the detection accuracy and specificity.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a primer probe set, i.e., a primer and a probe for chlamydia pneumoniae and chlamydia trachomatis markers, which can simultaneously detect chlamydia pneumoniae and chlamydia trachomatis DNAs.
The second purpose of the invention is to provide a kit which can simultaneously detect DNA of Chlamydia pneumoniae and Chlamydia trachomatis; so as to relieve the technical problem of the prior art that the detection specificity of the chlamydia is not strong.
The third purpose of the invention is to provide the application of the primer probe group capable of simultaneously detecting the DNA of the Chlamydia pneumoniae and the Chlamydia trachomatis in the kit for detecting the DNA of the Chlamydia pneumoniae and the Chlamydia trachomatis.
The primer probe combination provided by the invention is applied to the preparation of the kit for detecting the chlamydia pneumoniae and the chlamydia trachomatis, so that the technical problem that the diagnosis and research on the chlamydia in the prior art are insufficient can be solved.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides a primer probe group, which is used for simultaneously detecting DNA of Chlamydia pneumoniae and Chlamydia trachomatis and comprises the primer probe group capable of simultaneously detecting DNA of Chlamydia pneumoniae and Chlamydia trachomatis;
the primer pair of the chlamydia pneumoniae and the chlamydia trachomatis comprises an upstream primer with a sequence shown as SEQ ID NO.1 and a downstream primer with a sequence shown as SEQ ID NO.2, and probe sequences of the chlamydia pneumoniae and the chlamydia trachomatis are shown as SEQ ID NO. 3.
Further, the primer probe set also comprises a housekeeping gene (IC) (BH) primer pair and a probe, wherein the housekeeping gene (IC) (BH) primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.4 and a downstream primer with a sequence shown as SEQ ID NO.5, and the probe sequence is shown as SEQ ID NO. 6.
Furthermore, FAM luminescent groups are marked at the 5 'ends of the Chlamydia pneumoniae and Chlamydia trachomatis probes, and fluorescence quenching groups BHQ1 are marked at the 3' ends; the 5 'end of the housekeeping gene probe is marked with a CY5 luminescent group, and the 3' end is marked with a fluorescence quenching group BHQ 2.
The invention also provides a kit for simultaneously detecting DNA of chlamydia pneumoniae and chlamydia trachomatis, which comprises the primer probe set, buffer solution, dNTPs and MgCl 2 DEPC water and Taq enzyme.
Further, the final concentration of the primer in the amplification system is 100-1000 nM; the final concentration of the probe in the amplification system is 50-500 nM. The primers refer to primer pairs of chlamydia pneumoniae and chlamydia trachomatis and housekeeping gene primer pairs; the probes refer to probes for Chlamydia pneumoniae and Chlamydia trachomatis and housekeeping gene probes.
Furthermore, the detection sample of the kit is a throat swab.
The invention also provides application of the primer probe set in a kit for simultaneously detecting the DNA of the chlamydia pneumoniae and the Chlamydia trachomatis.
The invention has the beneficial effects that:
1. the probe used by the invention is a common segment of chlamydia trachomatis and chlamydia pneumoniae, and has higher specificity.
2. Compared with the traditional chlamydia detection kit, the kit only uses one probe, reduces the components of the kit, simplifies the operation and saves the cost.
3. The invention has the advantages of high sensitivity, good specificity and the like, and can effectively avoid the problems of false positive, false negative and the like.
Drawings
FIG. 1 is a graph of primer probe set performance verification calculated against Chlamydia pneumoniae standard;
FIG. 2 is a graph of primer probe set performance verification back calculated Chlamydia trachomatis standard;
FIG. 3 is a QPCR amplification graph of Chlamydia pneumoniae after extraction by the kit;
FIG. 4 is a QPCR amplification curve chart of Chlamydia trachomatis extracted by the kit;
FIG. 5 is a graph of specific experimental QPCR amplification.
Detailed Description
The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
The kit consists of primer probe mixed liquor, RT-PCR buffer, mixed enzyme liquid, a positive quality control product and RNase-free water; RT-PCR buffer includes PCR buffer, dATP, dUTP, dCTP, dGTP and MgCl 2 (ii) a Hot Start Taq enzyme; the positive quality control product contains plasmid of amplified gene sequence of chlamydia.
The primer pair of the chlamydia pneumoniae and the chlamydia trachomatis comprises an upstream primer with a sequence shown as SEQ ID NO.1 and a downstream primer with a sequence shown as SEQ ID NO.2, and the probe sequences of the chlamydia pneumoniae and the chlamydia trachomatis are shown as SEQ ID NO. 3.
The housekeeping gene (IC) (BH) primer pair comprises an upstream primer with a sequence shown as SEQ ID NO.4 and a downstream primer with a sequence shown as SEQ ID NO.5, and the housekeeping gene probe sequence is shown as SEQ ID NO. 6.
SEQ ID NO.1：5’ATTTCCGATTGCAATATGGAAGAAG 3’；
SEQ ID NO.2：5’CTGTGCTGATGCGTCTCAAAGA 3’；
SEQ ID NO.3：5’FAM-CGTTTTGATGTCAATGTCTCCGTACGC-BHQ1 3’；
SEQ ID NO.4：5’TCTGGCACCACACCTTCTACAA 3’；
SEQ ID NO.5：5’GGATAGCACAGCCTGGATAGCA 3’；
SEQ ID NO.6：5’FAM-AGGAGCACCCCGTGCTGCTGAC-BHQ2 3’。
The final concentration of the primer in the amplification system is 100-1000 nM; the final concentration of the probe in the amplification system is 50-500 nM.
The reaction system of the kit is 30 mu L, and specifically comprises: nucleic acid template 7. mu. L, RT-PCR buffer 20. mu.L, mixed enzyme solution 1. mu.L and primer-probe mixture 2. mu.L.
Operation of the kit and determination of the result
(1) Adding 7 mu L of each sample extraction template, RNase-free water and positive quality control material into different PCR reaction tubes, adding 20 mu L of RT-PCR buffer, 1 mu L of Hot Start Taq enzyme and 2 mu L of primer probe mixed solution into each tube to prepare a system, covering a tube cover, and placing the system into a fluorescent quantitative PCR instrument for fluorescent PCR detection.
(2) The conditions for PCR amplification reaction in the instrument were set as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 10 s; annealing at 60 ℃ for 30 s; 40 cycles.
(3) After the reaction is finished, setting the base line as automatic adjustment, and analyzing the detection result according to the amplification curve graph and the Ct value.
(4) And (3) judging the effectiveness:
the Ct value detected by the RNase-free water is Undet or 40, and the Ct value detected by the positive quality control product is less than or equal to 38, otherwise, the experiment is regarded as invalid;
(5) judging the result:
the Ct value of the sample detection hole is Undet or 40, the sample result is judged to be negative, the sample DNA/RNA extraction fails, the sample to be detected does not contain DNA/RNA or the content is lower than the detection limit;
the Ct value of the sample detection hole is less than or equal to 38, the sample result is judged to be positive to the corresponding pathogen, and the sample detection is successful;
and (4) the Ct value of the sample detection hole is 38-40, the retest is required once, and if the Ct value is still 38-40, the sample detection hole is judged to be negative.
And (3) verifying the performance of the primer probe:
respectively detecting positive samples of the chlamydia pneumoniae and the chlamydia trachomatis, and diluting the samples by 5 times in a gradient manner for 5 concentrations;
the concentrations are respectively 1 × 10 8 copies/ml、2×10 7 copies/ml、4×10 6 copies/ml、8×10 5 copies/ml、1.6×10 5 copies/ml .
The amplification efficiency of the chlamydia pneumoniae can reach 84.38%, and the amplification efficiency of the chlamydia trachomatis can reach 94.19%;
the calculation formula of the amplification efficiency is as follows: the amplification efficiency is (10^ (-1/K) -1) × 100%, where K is the slope in FIGS. 1 and 2.
The graph of the standard curve calculated after the experiment is shown in figures 1 and 2.
From FIGS. 1 and 2, the standard curve R of the primer probe can be seen 2 More than 0.99, and the performance meets the requirement.
And (3) sensitivity verification:
the kit has high detection sensitivity, and can detect 1 × 10 3 chlamydia trachomatis and chlamydia pneumoniae in copies/ml. 6 linear sensitivity samples were taken at 1X 10 concentrations 8 copies/ml、1×10 7 copies/ml、1×10 6 copies/ml、1×10 5 copies/ml、1×10 4 copies/ml、1×10 3 copies/ml、1×10 2 The copies/ml are subjected to 3 times of repeated experiments, and the lowest detection lower limit reaches 1 multiplied by 10 after verification experiments 3 copies/ml, the detection results are shown in FIGS. 3 and 4.
And (3) specificity verification:
the test results show that the kit has strong detection specificity for the chlamydia trachomatis and the chlamydia pneumoniae, does not have cross reaction with nucleic acids of other pathogens, detects 6 common clinical samples, and the results are shown in figure 5.
Table 1 summarizes the data statistics of the sensitivity verification experiment, and the experiment shows that the lower limit of the detection of the kit can reach 5 × 10 3 copies/ml, the kit has very good sensitivity.
Table 2 summarizes the data statistics of the specificity verification experiment, and the experiment shows that the kit has good specificity.
TABLE 1 sensitivity
Figure BDA0003716352080000051
TABLE 2 specificity
Figure BDA0003716352080000052
It should be noted that the above-mentioned contents only illustrate the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and it will be apparent to those skilled in the art that several modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments fall within the protection scope of the claims of the present invention.
Sequence listing
<110> Aike Rui Te biomedical science and technology (Nanjing) Co., Ltd
<120> kit for simultaneously detecting DNA of Chlamydia pneumoniae and Chlamydia trachomatis and application thereof
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cgttttgatg tcaatgtctc cgtacgc 27
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Claims (7)

1. A primer probe group is used for simultaneously detecting DNA of Chlamydia pneumoniae and Chlamydia trachomatis, and comprises the primer probe group capable of simultaneously detecting DNA of Chlamydia pneumoniae and Chlamydia trachomatis;
wherein, the primer pair sequence of the chlamydia pneumoniae and the chlamydia trachomatis is shown as SEQ ID NO.1-2, and the probe sequence of the chlamydia pneumoniae and the chlamydia trachomatis is shown as SEQ ID NO. 3.
2. The primer probe set of claim 1, further comprising a housekeeping gene primer pair and a housekeeping gene probe, wherein the sequence of the housekeeping gene primer pair is shown in SEQ ID No.4-5, and the sequence of the housekeeping gene probe is shown in SEQ ID No. 6.
3. The primer probe set of claim 2, wherein the 5 'end of the Chlamydia pneumoniae or Chlamydia trachomatis probe is labeled with FAM luminescent group, and the 3' end is labeled with fluorescence quenching group BHQ 1; the 5 'end of the housekeeping gene probe is marked with a CY5 luminescent group, and the 3' end is marked with a fluorescence quenching group BHQ 2.
4. A kit for simultaneously detecting DNA of Chlamydia pneumoniae and Chlamydia trachomatis, comprising the primer probe set according to any one of claims 1 to 3, a buffer solution, dNTPs, MgCl 2 DEPC water and Taq enzyme.
5. The kit for simultaneously detecting Chlamydia pneumoniae and Chlamydia trachomatis DNA according to claim 4, wherein the final concentration of the primers in the primer probe set in the amplification system is 100-1000 nM; the final concentration of the probe in the primer probe group in the amplification system is 50-500 nM.
6. The kit for simultaneously detecting the DNA of the Chlamydia pneumoniae and the Chlamydia trachomatis according to claim 4, wherein the detection sample of the kit is a throat swab.
7. Use of a primer probe set according to any one of claims 1 to 3 in a kit for the simultaneous detection of DNA from Chlamydia pneumoniae and Chlamydia trachomatis.
CN202210737325.2A 2022-06-27 2022-06-27 Kit for simultaneously detecting DNA of chlamydia pneumoniae and Chlamydia trachomatis and application thereof Pending CN114921575A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106086198A (en) * 2016-06-29 2016-11-09 湖州市中心医院 Multiple fluorescence quantitative PCR detects primer and the probe of mycoplasma pneumoniae, Chlamydia pneumoniae and chlamydia trachomatis simultaneously
CN112301169A (en) * 2020-12-30 2021-02-02 爱科睿特生物医疗科技(南京)有限公司 Primer group, probe group and kit for synchronously detecting pathogens related to multiple genital tract infections

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106086198A (en) * 2016-06-29 2016-11-09 湖州市中心医院 Multiple fluorescence quantitative PCR detects primer and the probe of mycoplasma pneumoniae, Chlamydia pneumoniae and chlamydia trachomatis simultaneously
CN112301169A (en) * 2020-12-30 2021-02-02 爱科睿特生物医疗科技(南京)有限公司 Primer group, probe group and kit for synchronously detecting pathogens related to multiple genital tract infections

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIRGIT KRAUSSE-OPATZ ET AL.: "Production of prostaglandin E2 in monocytes stimulated in vitro by Chlamydia trachomatis, Chlamydophila pneumoniae, and Mycoplasma fermentans", MICROB PATHOG., vol. 37, no. 3, pages 155 - 161, XP004537587, DOI: 10.1016/j.micpath.2004.06.007 *
冯艳铭;吴逸明;吴拥军;马俊芬;李振勇;邵大哓;刘丁;: "TaqMan PCR技术同步检测沙眼衣原体及肺炎衣原体的DNA", 临床检验杂志, no. 03, pages 70 - 71 *
程永婷;贾晓晖;马良;贾天军;: "肺炎衣原体Taqman探针实时荧光定量PCR检测法", 临床检验杂志, no. 05, pages 343 - 346 *

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Application publication date: 20220819