CN111549160A - Primer group and kit for detecting leishmania and application of primer group and kit - Google Patents
Primer group and kit for detecting leishmania and application of primer group and kit Download PDFInfo
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Abstract
The invention discloses a primer group and a kit for detecting leishmania and application thereof, belonging to the technical field of biology. The primer group for detecting leishmania comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as a sequence table SEQ ID NO. 1; the nucleotide sequence of the downstream primer is shown in a sequence table SEQ ID NO. 2. By using the kit containing the primer group and the TaqMan probe with the nucleotide sequence shown in the sequence table SEQ ID NO. 3, whether Leishmania DNA exists in a human or animal bone marrow specimen can be detected, so that the laboratory diagnosis efficiency of kala-azar etiology can be improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group and a kit for detecting leishmania and application thereof.
Background
Kala-azar (also known as visceral leishmaniasis) is a common disease of both humans and animals with major symptoms of fever, progressive splenomegaly, and trilineage cell loss caused by leishmania. Is one of five parasitic diseases which harm the health of people in China.
The experimental diagnosis of the kala-azar can be realized by serological detection of a Leishmania antibody, a marrow puncture smear and microscopic search of the 'Ledu body'. However, the existing protozoan antibody detection reagent needs to be imported, is not produced in China, needs certain experience in microscopic examination, and has higher misdiagnosis and missed diagnosis rate.
The gene diagnosis can detect trace pathogen nucleic acid fragments, has the characteristics of rapidness, sensitivity and high specificity, and is increasingly applied to the detection of pathogens. Leishmania infection is localized to macrophages and a definitive diagnosis of etiology can be rapidly obtained by detecting Leishmania gene fragments in the patient's bone marrow or blood DNA. The conventional method for diagnosing leishmania genes comprises a Polymerase Chain Reaction (PCR) method, but the method needs steps of gel electrophoresis, ultraviolet development, photographing and the like, and is long in time consumption. In addition, there is a fluorescence quantitative PCR method using SYBG dye method, but this method detects the fluorescence signal as non-specificity, and needs to use the melting curve to judge, and the melting curves of different instruments have certain difference, so it is difficult to standardize, and has certain defects, and the gDNA composition of clinical specimen is complex, and the non-specific amplification is easy to occur, so the false positive appears.
Disclosure of Invention
The present invention is intended to solve the problems of the background art mentioned above by providing a primer set for detecting Leishmania.
In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:
a primer group for detecting Leishmania comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as a sequence table SEQ ID NO. 1; the nucleotide sequence of the downstream primer is shown in a sequence table SEQ ID NO. 2.
Another objective of the embodiments of the present invention is to provide a kit comprising the primer set.
As another preferable scheme of the embodiment of the invention, the kit further comprises a TaqMan probe; the nucleotide sequence of the TaqMan probe is shown in a sequence table SEQ ID NO. 3.
As another preferred scheme of the embodiment of the invention, two ends of the TaqMan probe are respectively connected with a reporter group and a quencher group; the reporter group is FAM (6-carboxyfluorescein) group, and the quenching group is Minor Groove Binding (MGB) group.
As another preferred embodiment of the present invention, the kit further comprises a positive plasmid standard; the preparation method of the positive plasmid standard substance comprises the following steps:
performing PCR amplification on the leishmania positive specimen by using the primer group to obtain a PCR product;
and cutting the PCR product, recovering and purifying, connecting to a vector, cloning to escherichia coli, and extracting plasmids to obtain the positive plasmid standard substance.
As another preferable scheme of the embodiment of the invention, the nucleotide sequence of the positive plasmid standard substance is shown in a sequence table SE Q ID NO. 4.
The embodiment of the invention also aims to provide application of the kit in preparing a kit for detecting the kala-azar.
Compared with the prior art, the embodiment of the invention has the beneficial effects that:
according to the primer group for detecting leishmania provided by the embodiment of the invention, by using the kit containing the primer group and the TaqMan probe with the nucleotide sequence shown in SEQ ID NO. 3 of the sequence table, whether leishmania DNA exists in a human or animal bone marrow specimen can be detected, and thus the laboratory diagnosis efficiency of kala-azar etiology can be improved.
Specifically, the kit provided by the embodiment of the invention can establish a fluorescence quantitative PCR detection method of Leishmania by taking the peculiar kinetochore ringlet of Leishmania as a target gene by using a TaqMan probe method. The whole detection time can be finished within 30-40 minutes, and the time is obviously reduced compared with that of a common PCR method; the total length of the upstream and downstream primers and the probe is 55bp, the length of an amplification product is 83bp, and compared with a fluorescent quantitative PCR method by an SYBG dye method, the specificity of detection can be effectively ensured; the scheme can also carry out absolute quantitative detection on the target nucleic acid fragment in the sample by establishing a standard curve and a regression equation, the lower detection limit can reach 26.98 copies/mu L, and 1 protozoon/mL can be detected. The method overcomes the defects that the common PCR method has long detection time and needs open-tube electrophoresis to easily cause product pollution, also avoids the influence of searching protozoan subjective factors under a mirror, detects leishmania nucleic acid fragments which are equal to the existence of pathogens, and provides a simple, convenient, sensitive and rapid detection method for clinical diagnosis of kala-azar and epidemiological investigation. In addition, the kit can also generate a positive result in the blood detection of the fever stage case of the kala-azar.
Drawings
FIG. 1 is a PCR amplification curve of positive plasmid standards at different concentrations.
FIG. 2 is a real-time fluorescence quantitative PCR standard regression curve of the kit provided by the embodiment of the invention in the detection of Leishmania.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This example provides a kit for detecting leishmania comprising a PCR premix, sterile nuclease-free double distilled water (ddH)2O), a primer group, a probe and a positive plasmid standard substance, wherein the PCR premix solution can adopt a 2 × TaqMan Fast qPCR Master Mix product sold by Shanghai bio (Shanghai) GmbH, the nucleotide sequence of an upstream primer is shown as a sequence table SEQ ID NO:1 and is specifically 5'-GCAGAAATCCCGTTCAAA-3', the nucleotide sequence of a downstream primer is shown as a sequence table SEQ ID NO:2 and is specifically 5'-GCCCTATTTTACACC AACC-3', the nucleotide sequence of the TaqMan probe is shown as a sequence table SEQ ID NO:3, and two ends of the TaqMan probe are respectively connected with a report group and a quenching group, wherein the report group is a FAM group, the quenching group is a MGB group, and the nucleotide sequence of the TaqMan probe is specifically 5 '-6 FAM-CGCCCCGGAGCCGATTTT-MGB-3'.
In addition, the preparation method of the positive plasmid standard substance comprises the following steps:
(1) and carrying out PCR amplification on the leishmania positive specimen by using the primer group to obtain a PCR product. Wherein, the nucleotide sequence of the positive plasmid standard substance is shown as a sequence table SEQ ID NO. 4, and specifically comprises the following components: GCAGAAATCCCGTTCAAAAATCGGCCAAAAATGCCAAAAATCGGCTCCGGGGCGGGAAACTG GGGGTTG GTGTAAAATAGGGC are provided.
(2) The PCR product was recovered and purified using a SanPrep column type DNA gel recovery kit (produced by Biotechnology Ltd., Shanghai), 1uL of the recovered product was taken, and 1uL of pESI-T vector and 1uL 10 × Enhancer (provided by san Biotechnology Ltd., Shanghai), ddH, were added to the recovered product2Supplementing O to 10uL, reacting at room temperature for 5min, adding 100 uL competent cell DH5 α, adding 500 uL LB culture medium (without antibiotic), shaking and culturing at 37 ℃ and 180rpm for 10min, taking 200 uL bacterial liquid, coating LB culture medium containing ampicillin resistance at 37 ℃ and culturing overnight, picking up single clone colony, extracting plasmid with QIAprep Spi nMumpirep Kit plasmid miniextraction Kit (Qiagen company), sequencing with universal primer (M13F: TGTAAAACGACGGCCAGT M13R: CAGGAAACAGCTATGACC), comparing Blast to obtain clone with leishmania dynamic matrix minicircle specific sequence as positive clone, measuring OD value with Nanoop photometer for positive plasmid, calculating copy number with Avogardro constant formula, and diluting to 10uL8Obtaining positive plasmid standard product by copies/ul.
The forward primer, the reverse primer and the probe may be present in the form of a solution, and the concentrations thereof may be set to 10. mu.M.
Example 2
This example provides a method for detecting leishmania using the kit provided in example 1 above, comprising the steps of:
(1) a DNA template was obtained by taking 200. mu.L of a bone marrow or Blood sample (the surface of a slide glass of which a bone marrow smear specimen was smeared with a sterile wet cotton swab, suspending the sample in 300. mu.L of sterile water by shaking, and then taking 200. mu.L of the sample), and extracting the DNA of the bone marrow sample using a DNeasy Blood & Tissue Kit (Cat No.69504) DNA extraction Kit manufactured by Qiagen according to a Blood Tissue or bacteria-based group extraction procedure.
(2) mu.L of the PCR master mix and 1. mu.L of the forward primer were each collected from the kit provided in example 11 μ L of downstream primer, 1 μ L of TaqMan probe and 1 μ L of the DNA template obtained above were mixed, followed by dd H2The amount of O was adjusted to 20. mu.L to obtain a reaction mixture.
(3) Placing the obtained reaction solution in a fluorescent PCR instrument for amplification reaction, and collecting a fluorescent signal; meanwhile, under the same conditions, a positive plasmid standard substance in the kit is used as a positive control, sterile double distilled water is used as a blank control, if the CT value of the positive control is less than 30 and the CT value of the blank control is more than or equal to 35, the CT value of the bone marrow sample is less than 35 and an obvious amplification curve exists, the bone marrow sample can be judged to be positive, and the existence of leishmania zoosome gene segments in the bone marrow sample is proved; if the CT value of the bone marrow sample is more than or equal to 35, the bone marrow sample can be judged to be negative, and the leishmania zoosome genes in the bone marrow sample are excluded. Wherein, the fluorescent PCR instrument is arranged as follows: the reporter group is FAM, the quenching group is MGB, and on an ABi series fluorescent PCR instrument, the quenching gene is selected from None and the fluorescent None is corrected; the PCR amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 5s and extension at 60 ℃ for 20s, and fluorescence signals were collected for 40 cycles.
Example 3
This example provides a PCR amplification experiment using the kit provided in example 1 above for different concentrations of positive plasmid standards, comprising the steps of:
(1) respectively diluting the positive plasmid standard substance to 107copies/μL、106copies/μL、105copies/μL、104co pies/μL、103copies/μL、102copies/. mu.L for use.
(2) Taking 1 μ L of different concentrations (10)2～108copies/. mu.L) was mixed with 10. mu.L of the PCR premix, 1. mu.L of the upstream primer, 1. mu.L of the downstream primer, and 1. mu.L of the TaqMan probe, and then ddH was added thereto2The total amount of O was adjusted to 20. mu.L, to obtain 7 groups of reaction solutions.
(3) The 7 groups of reaction solutions obtained above were placed in a fluorescence PCR instrument for amplification reaction, and fluorescence signals were collected to obtain 7 groups of PCR amplification curves, as shown in FIG. 1. WhereinThe fluorescent PCR instrument was set up as follows: the reporter group is FAM, the quenching group is MGB, and on an ABi series fluorescent PCR instrument, the quenching gene is selected from None and the fluorescent None is corrected; the PCR amplification reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 5s and extension at 60 ℃ for 20s, and fluorescence signals were collected for 40 cycles. In FIG. 1, A to H from left to right are 10 in this order8copies/μL、107copies/μL、106copies/μL、105copies/μL、104copies/μL、103copies/μL、102As can be seen from the PCR amplification curve corresponding to the copies/. mu.L positive plasmid standard, the detection lower limit of the kit provided by the embodiment of the invention is lower.
In addition, according to the amplification experiment of the positive plasmid standard products with different concentrations, a real-time fluorescence quantitative PCR standard regression curve of the kit for detecting leishmania can be calculated, as shown in the attached figure 2, the standard regression curve equation is Y ═ 41.926-3.086X, the amplification Effect (EF) is 110.894%, and the correlation coefficient R is2Was 0.988. The presence of Leishmania in the sample to be tested can be quantitatively analyzed according to the standard regression curve equation.
In conclusion, the kit provided by the embodiment of the invention can be applied to detection of the etiology of the kala-azar, and can greatly improve the laboratory diagnosis efficiency of the etiology of the kala-azar. The positive rate of myeloscopy of the existing kala-azar cases is about 70% generally, but the positive rate of myeloscopy of the kala-azar cases can be improved to 100% by using the kit provided by the embodiment of the invention, and the detection time can be shortened to be within 1 hour. The kit can also detect a positive result on the blood of a case in the fever period of the kala-azar.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
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Claims (7)
1. A primer set for detecting leishmania, wherein the primer set comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown as a sequence table SEQ ID NO. 1; the nucleotide sequence of the downstream primer is shown in a sequence table SEQ ID NO. 2.
2. A kit comprising the primer set of claim 1.
3. The kit of claim 2, wherein the kit further comprises a TaqMan probe; the nucleotide sequence of the TaqMan probe is shown in a sequence table SEQ ID NO. 3.
4. The kit of claim 3, wherein two ends of the TaqMan probe are respectively connected with a reporter group and a quencher group; the reporter group is FAM group, and the quencher group is MGB group.
5. The kit of claim 2, further comprising a positive plasmid standard; the preparation method of the positive plasmid standard substance comprises the following steps:
performing PCR amplification on the leishmania positive specimen by using the primer group to obtain a PCR product;
and cutting the PCR product, recovering and purifying, connecting to a vector, cloning to escherichia coli, and extracting plasmids to obtain the positive plasmid standard substance.
6. The kit according to claim 5, wherein the nucleotide sequence of the positive plasmid standard is shown in SEQ ID NO. 4 of the sequence table.
7. Use of the kit according to any one of claims 2 to 6 for the preparation of a kit for detecting kala-azar.
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| CN1973049A (en) * | 2003-12-23 | 2007-05-30 | 全印度医疗科学学会 | Oligonucleotides for detection of leishmaniasis and methods thereof |
| WO2014111207A1 (en) * | 2013-01-18 | 2014-07-24 | Philipps-Universität Marburg | Diagnosis of leishmania infection |
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| Title |
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| 白雪飞等: "PCR技术在利什曼原虫无症状感染诊断中的应用研究进展", 《中国病原生物学杂志》 * |
| 谢晖等, 西安交通大学出版社 * |
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