CN107058502B - Primer pair and kit for detecting mouse trypanosomes - Google Patents
Primer pair and kit for detecting mouse trypanosomes Download PDFInfo
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Abstract
The invention discloses a primer pair and a kit for detecting trypanosomes of mice; by comparing macrocyclic sequences of the mouse trypanosome and other similar trypanosomes, a pair of primer pairs TM2-F and TM2-R capable of specifically detecting the mouse trypanosome is screened, wherein the sequences of the primer pairs are shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; the primer pair can specifically amplify partial fragments of the mouse trypanosomes, has high sensitivity, and can detect a sample containing only 10 trypanosomes or a DNA sample of 1 ng; the primer pair can be used for quickly and accurately separating the trypanosoma from other trypanosomes and leishmania (L.)Leishmania amazonesis) The method does not need expensive microscope and professional etiology knowledge, is quick and convenient to detect, can be used for detecting early infection, is suitable for detecting animal trypanosome mixed infection, is beneficial to developing epidemiological investigation and is possibly suitable for detecting the trypanosome of human infected mice.
Description
Technical Field
The invention relates to the technical field of parasite molecule detection, and particularly relates to a primer pair and a kit for detecting mouse trypanosomes.
Background
In nature, Trypanosoma micranthum: (Trypanosoma musculi) Is a parasitic protozoa distributed globally and spread among mammals by means of vector insect fleas, belonging to the sub genus (A), (B), (CSubgenus Herpetosoma) For the detection of mouse trypanosomes, the prior art develops a plurality of modern detection means. For example, the ITS technique is defective, so that only other subgenera can be distinguished, and although the wide distribution of protozoa is known, epidemiological data is outdated due to public neglect, and epidemiological investigation is necessary to evaluate the risk of infecting people.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the problems in the prior art and provide a primer for detecting the mouse trypanosomes.
The second purpose of the invention is to provide a kit containing the primer.
The purpose of the invention is realized by the following technical scheme:
a primer pair TM2-F and TM2-R for detecting mouse trypanosomes, wherein the sequence of the primer pair is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The invention compares the genes of the mouse trypanosome and other similar trypanosomes, and screens out a pair of primer pairs capable of specifically detecting the mouse trypanosome through primer design.
Therefore, the invention also provides a kit containing the primer pair.
Preferably, the kit also contains PCR amplification reaction reagents; the PCR amplification reaction reagent is a reagent which enables the sample DNA to perform PCR amplification reaction under the action of the primer pair, and conventional PCR amplification reaction reagents in the prior art can be used in the present invention.
The PCR amplification reaction reagents may be commercially available reagents, such as PrimerSTAR Max Premix.
Preferably, if the sample is a DNA sample, the PCR amplification reaction system of the kit is as follows: 12.5. mu.l of PCR reaction reagent, primer TM2-F and primer TM2-R, 100ng of sample DNA, and sterile double distilled water were made up to 25. mu.l, and the final concentrations of primer TM2-F and primer TM2-R in the reaction system were 6.25pmol/L, respectively.
Preferably, if the sample is a blood sample, the PCR amplification reaction system of the kit is: mu.l of PCR reaction reagent, primer TM2-F and primer TM2-R, 10. mu.l of sample hemolysate, sterile double distilled water was added to make up to 25. mu.l, and the final concentrations of primer TM2-F and primer TM2-R in the reaction system were 6.25pmol/L, respectively.
Preferably, the PCR amplification reaction conditions of the kit are as follows: 30 reaction cycles: denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 15s, and extension at 72 ℃ for 8 s.
The method for detecting the mouse trypanosomes by utilizing the kit through PCR comprises the following steps: (1) extracting sample DNA or sample blood; (2) adding a PCR amplification reaction reagent into the sample DNA or the sample blood for PCR amplification, and judging the result: if a band with the size of 1313 bp is amplified, the detected sample contains the mouse trypanosome, otherwise, the detected sample does not contain the mouse trypanosome.
Compared with the prior art, the invention has the following beneficial effects:
the invention relates to a primer designed by utilizing a large ring of kDNA (kinetoplast DNA), namely, a pair of primer pairs TM2-F and TM2-R capable of specifically detecting the mouse trypanosome is screened out by comparing the large ring sequences of the mouse trypanosome and other similar trypanosomes, wherein the sequences of the primer pairs are shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification; the primer pair can specifically amplify partial fragments of the mouse trypanosomes, has high sensitivity, and can detect a sample containing only 10 trypanosomes or a DNA sample of 1 ng; the primer pair can be used for quickly and accurately separating the trypanosoma from other trypanosomes and leishmania (L.)Leishmania amazonesis) The method is characterized in that PCR detection is directly carried out based on tail blood of mice infected with trypanosomes, so that the detection operability is improved, the detection is fast and convenient, the method can be used for detecting early infection, is suitable for detecting mixed infection of animal trypanosomes, is beneficial to epidemiological investigation and is possibly suitable for detecting human infected mice trypanosomes.
Drawings
FIG. 1 shows the result of optimizing the reaction temperature of PCR detection of mouse trypanosomes by TM 2; in the figure: lane M: DL2000marker, lanes 1 to 6 are 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃ and 60 ℃ respectively, and lane N is a negative control.
FIG. 2 shows the result of optimizing the reaction temperature of PCR detection of mouse trypanosomes by TM 1; in the figure: lane M: DL2000marker, lanes 1 to 6 are 50 ℃, 52 ℃, 54 ℃, 56 ℃, 58 ℃ and 60 ℃ respectively, and lane N is a negative control.
FIG. 3 shows the result of PCR detection of mouse trypanosoma by TM 2; in the figure: lane M: DL2000marker, 3 kinds of lanes 1-3T. musculi(Trypanosoma micranthum) 4-6 of 3 speciesT. lewisi(Trypanosoma ludwigii) 7-9 are 3T. brucei(Trypanosoma brucei), lane 10T. evansiTrypanosoma evansi, lanes 11-13 are in 3T. equiperdum(Trypanosoma vernalium), lane 14T. cruziT.kupffer, lane 15 isL. amazonensis(Leishmania), lane N as a negative control.
FIG. 4 shows the result of PCR detection of mouse trypanosoma by TM 1; in the figure: lane M: DL2000marker, 3 kinds of lanes 1-3T. musculi(Trypanosoma micranthum) 4-6 of 3 speciesT. lewisi(Trypanosoma ludwigii) 7-9 are 3T. brucei(Trypanosoma brucei), lane 10T. evansiTrypanosoma evansi, lanes 11-13 are in 3T. equiperdum(Trypanosoma vernalium), lane 14T. cruziT.kupffer, lane 15 isL. amazonensis(Leishmania), lane N as a negative control.
FIG. 5 shows the PCR detection sensitivity of TM2 on mouse trypanosoma DNA; in the figure: lane M: DL2000marker, lanes 1-6 are 200 ng, 100ng, 50 ng, 10 ng, 1ng, 0.1 ng, respectively, lane N is negative control.
FIG. 6 shows the PCR detection sensitivity of TM1 on mouse trypanosoma DNA; in the figure: lane M: DL2000marker, lanes 1-6 are 200 ng, 100ng, 50 ng, 10 ng, 1ng, 0.1 ng, respectively, lane N is negative control.
FIG. 7 shows the PCR detection sensitivity of TM2 on mouse trypanosoma blood; in the figure: lane M: DL2000marker, lanes 1-5 are 10 respectively4A trypanosoma, 103A trypanosoma, 102One trypanosome, 10 trypanosomes, 1 trypanosome, swimmingLane N is a negative control.
FIG. 8 shows the PCR detection sensitivity of TM1 on mouse trypanosoma blood; in the figure: lane M: DL2000marker, lanes 1-5 are 10 respectively4A trypanosoma, 103A trypanosoma, 102Trypanosomes, 10 trypanosomes, 1 trypanosome, lane N is negative control.
FIG. 9 is a flow chart of PCR detection of blood samples.
FIG. 10 shows the target fragment amplified by PCR, and the underlined English letters indicate the primer binding regions.
Detailed Description
The invention is further illustrated by the following figures and specific examples in conjunction with the description. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures, in which specific conditions are not indicated in the examples below, are generally carried out according to conditions conventional in the art or as recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art.
Trypanosoma micranthum (Trypanosoma micranthum) in miceT. mucsuliFRA) extraction of total DNA:
(1) adding SNET buffer solution (adding proteinase K into the SNET buffer solution, wherein the final concentration of the proteinase K is 100 mu g/ml), and digesting for 3-5 h at 56 ℃ in a metal bath;
(2) taking out the sample in the step (1), adding RNA enzyme, and digesting for 0.5 h at 37 ℃ in a metal bath;
(3) removing the sample in the step (2), and adding a mixture of 25: 24: 1 phenol: chloroform: shaking and uniformly mixing isoamyl alcohol at room temperature to form emulsion, and standing for 2-5 min;
(4) centrifuging the mixed solution obtained in the step (3) at 12000 rpm for 10 min, and transferring the upper aqueous phase into a new centrifugal tube;
(5) adding equal volume of isopropanol into the water phase in the step (4) to precipitate DNA, centrifuging at 12000 rpm for 5 minutes, and then removing supernatant;
(6) washing the precipitate collected in the step (5) with 300 mul of precooled 70% ethanol, then centrifuging at 12000 rpm for 5 min at a high speed, removing the supernatant, and volatilizing the ethanol at room temperature;
(7) DNA samples were lysed with 50. mu.l TE to determine the DNA content.
Example 1 primer design
Mixing mouse trypanosoma (KY 426815) and Trypanosoma ludwigii (A. reum.) (T. lewisiCPO02, KR 072974), trypanosoma cruzi (GenBank: FJ 203996.1; GenBank: DQ 343646.1; GenBank: DQ 343645.1), and the primer sequences are shown in Table 1; wherein, Tubulin is an internal reference gene.
EXAMPLE 2 determination of temperature of PCR detection System
(1) Establishing a PCR amplification reaction system: 12.5. mu.l of PrimerSTAR Max Premix (TAKARA, Japan), 6.25pmol/L of forward and reverse primers, 100ng of the extracted DNA, and sterile double distilled water to make up to 25. mu.l, wherein the negative control was sterile double distilled water.
(2) PCR amplification reaction conditions: 30 reaction cycles: denaturation at 98 ℃ for 10s, (50-60 ℃) annealing for 15s, and extension at 72 ℃ for 8 s.
(3) And (2) analyzing amplification products, namely detecting the amplification products by agarose gel electrophoresis, taking 5 mu l of 25 mu l of final reaction products of polymerase chain reaction PCR amplification, adding 6 × loading buffer, spotting into 1% agarose gel containing GOLD VIEW dye, carrying out 220 v electrophoresis for 20 min, imaging and observing results on a gel phase formation system, selecting a reaction temperature of 52 ℃ for amplification of TM2 at 50-54 ℃ without basically changing the brightness of bands (figure 1), and amplifying target bands at 50-60 ℃ for amplification of TM1 (figure 2).
Example 3 PCR detection method specificity experiment
PCR amplification was performed according to the method of example 2.
(1) Establishing a PCR amplification reaction system: 12.5 μ L PrimerSTAR Max Premix (TAKARA, Japan), 6.25pmol/L primer TM2-F, 6.25pmol/L primer TM2-R (or 6.25pmol/L primer TM1-F, 6.25pmol/L primer TM 1-R), 100ng of DNA (15 samples), sterile double distilled water to make up to 25 μ L, with the negative control being sterile double distilled water.
(2) PCR amplification reaction conditions: 30 reaction cycles: denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 15s, and extension at 72 ℃ for 8 s.
(3) Analyzing the amplified product by agarose gel electrophoresis, taking 5 mul of the final reaction product of 25 mul of PCR amplification, adding 6 × loading buffer, spotting into 1% agarose gel containing GOLD VIEW dye, performing 220 v electrophoresis for 20 min, and imaging and observing the result on a gel phase system, wherein TM2 only can amplify the amplified productT. musculiPartial region (1313 bp) of (1) as shown in FIG. 3; the two pairs of primers have good specificity and do not have cross reaction with DNA of other species; TM1 was also only amplifiedT. musculiPartial region (fig. 4).
Example 4 PCR detection method sensitivity test (DNA)
PCR amplification was carried out according to the method of example 2, wherein 1. mu.l of DNA at various concentrations (200 ng/. mu.l, 100 ng/. mu.l, 50 ng/. mu.l, 10 ng/. mu.l, 1 ng/. mu.l, 0.1 ng/. mu.l) was added to 25. mu.l of the system (the amplification conditions of PCR were the same as in example 3).
The analysis of the amplification products was carried out according to the agarose gel electrophoresis method of example 2, and 1ng of DNA was detected as the lowest by TM2, as shown in FIG. 5; TM1 detected a minimum of 1ng of DNA, as shown in FIG. 6.
Example 5 PCR detection method sensitivity test (blood sample)
Collecting 1 ml of mouse tail blood of Kunming infected with mouse trypanosome in a small centrifuge tube, and diluting with mouse tail blood of non-inoculated trypanosome step by step respectively;
first, saponin treating tail blood to obtain blood dissolving liquid
(1) Sucking 5 mul of blood sample to be detected into a centrifuge tube;
(2) adding 500. mu.l of Saponin lysis buffer (0.22% NaCl, 0.015% Saponin, 1 mM EDTA) to the sample to be tested of step (1);
(3) centrifuging the mixed solution obtained in the step (2) for 1min at 12000 g, and removing the supernatant;
(4) the pellet from step (3) was washed three times with Saponin lysine buffer and resuspended in 100. mu.l of 1 XPCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.0, 0.5% Tween 20, 100. mu.g of protease Kper ml);
(5) placing the mixed solution in the step (4) in a metal constant temperature of 56 ℃ for enzymolysis reaction for 60 min;
(6) placing the mixed solution obtained in the step (5) in a metal constant temperature enzyme denaturation inactivation reaction at 95 ℃ for 5 min;
(7) and (4) centrifuging the mixed solution in the step (6) for 1min by using 12000 g to obtain a blood dissolved solution for PCR reaction.
Secondly, establishing a PCR amplification reaction system
12.5. mu.l PrimerSTAR Max Premix (TAKARA, Japan), primer TM2-F at a final concentration of 6.25pmol/L and primer TM2-R at a final concentration of 6.25pmol/L, 10. mu.l hemolysate (10. mu.l)3Trypanosoma/μ l, 102Each trypanosome/μ l, 10 trypanosomes/μ l, 1 trypanosome/μ l, 0.1 trypanosome/μ l), and sterile double distilled water is filled to 25 μ l, wherein the negative control is sterile double distilled water.
12.5. mu.l PrimerSTAR Max Premix (TAKARA, Japan), primer TM1-F at a final concentration of 6.25pmol/L and primer TM1-R at a final concentration of 6.25pmol/L, 10. mu.l hemolysate (10. mu.l)3/μl、102Mu.l, 10/mu.l, 1/mu.l, 0.1/mu.l), sterile double distilled water was replenished to 25 mu.l, wherein the negative control was sterile double distilled water.
PCR amplification reaction conditions: the same as in example 3.
Analysis of amplification products, namely taking 5. mu.l of 25. mu.l of final reaction product of PCR amplification, adding 6 × loading buffer, spotting into 1% agarose gel containing GOLD VIEW dye, carrying out 220 v electrophoresis for 20 min, and imaging and observing results on a gel phase forming system, wherein the results show that TM2 can detect blood samples containing 10 trypanosomes at minimum (figure 7), and TM1 can detect blood samples containing 10 trypanosomes at minimum (figure 7)4In blood samples of trypanosomes (fig. 8), the detection sensitivity of TM2 primer was significantly higher than that of TM1 primer.
In practical application, the detection sensitivity of TM1 is comparable to that of microscopic examination, so that the TM2 primer is suitable for detecting trypanosomes in early infection stage.
Example 6 detection kit containing primer TM2
The reagent kit comprises the following components: primers TM2-F and TM 2-R; PCR reagents such as PrimerSTAR Max Premix.
SEQUENCE LISTING
<110> Zhongshan university
<120> primer pair and kit for detecting mouse trypanosomes
<130>
<160>6
<170>PatentIn version 3.3
<210>1
<211>18
<212>DNA
<213>TM2-F
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tgggtttcca aatcctaa 18
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<212>DNA
<213>TM2-R
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gggagataaa gggatttc 18
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<212>DNA
<213>TM1-F
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taacgcaaag ttccaaatag 20
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<212>DNA
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gatgtaagag tagagttgga gg 22
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<213>Tubulin F
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ctggcttcaa gtgcggtatc aa 22
<210>6
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<213>Tubulin R
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gtactcctcc acatcctcct cg 22
Claims (6)
1. A primer pair TM2-F and TM2-R for detecting mouse trypanosomes, wherein the sequence of the primer pair is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
2. A kit comprising the primer set according to claim 1.
3. The kit of claim 2, wherein the kit further comprises a PCR reaction premix.
4. The kit according to claim 3, wherein the PCR amplification reaction system of the kit is: 12.5. mu.l of PCR reaction premix, primer TM2-F and primer TM2-R, 100ng of sample DNA, and sterile double distilled water to 25. mu.l; wherein the final concentrations of the primer TM2-F and the primer TM2-R in the reaction system are respectively 6.25 pmol/L.
5. The kit according to claim 3, wherein the PCR amplification reaction system of the kit is: 12.5 mul of PCR reaction premix, primer TM2-F and primer TM2-R, 10 mul of sample blood dissolving solution, and sterile double distilled water to make up to 25 mul; wherein the final concentrations of the primer TM2-F and the primer TM2-R in the reaction system are respectively 6.25 pmol/L.
6. The kit according to any one of claims 3 to 5, wherein the PCR amplification reaction conditions of the kit are as follows: 30 reaction cycles: denaturation at 98 ℃ for 10s, annealing at 52 ℃ for 15s, and extension at 72 ℃ for 8 s.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191324A (en) * | 2011-04-14 | 2011-09-21 | 吉林大学 | Trypanosoma evansi and trypanosoma lewisi dual-polymerase chain reaction (PCR) assay kit and preparation method thereof |
| CN106047993A (en) * | 2016-04-20 | 2016-10-26 | 金弗康生物科技(上海)有限公司 | Molecular markers for five important pathogens and application thereof |
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- 2017-02-15 CN CN201710081625.9A patent/CN107058502B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191324A (en) * | 2011-04-14 | 2011-09-21 | 吉林大学 | Trypanosoma evansi and trypanosoma lewisi dual-polymerase chain reaction (PCR) assay kit and preparation method thereof |
| CN106047993A (en) * | 2016-04-20 | 2016-10-26 | 金弗康生物科技(上海)有限公司 | Molecular markers for five important pathogens and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA;Desquesnes M等;《INTERNATIONAL JOURNAL FOR PARASITOLOGY》;20010501;第31卷(第5-6期);第610-614页 * |
| PCR identification of Trypanosoma lewisi, a common parasite of laboratory rats;M Desquesnes等;《Kinetoplastid Biol Dis》;20020529;第1卷(第1期);第2号 * |
| PCR-based identification of Trypanosoma lewisi and Trypanosoma musculi using maxicircle kinetoplast DNA;Hong XK等;《ACTA TROPICA》;20170731;第171卷;第207-212页 * |
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