CN107904322B - Specific primer for detecting trypanosoma protozoa, detection method and application - Google Patents
Specific primer for detecting trypanosoma protozoa, detection method and application Download PDFInfo
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Abstract
The invention discloses a group of specific primers for detecting trypanosoma protozoa, which have the following sequences: the upstream primer is as follows: 5' -CCTGATGAAAGGTGCAATG (SEQ ID NO:1) or a complementary sequence thereof; the downstream primer is: 5' -CGTTTTCGCCTTCTTGTGGA (SEQ ID NO:2) or the complement thereof. The invention also discloses a method for specifically detecting trypanosoma protozoa, which comprises the following steps: (1) extracting the genomic DNA of a target sample; (2) taking the genomic DNA of the target sample extracted in the step (1) as a template, and carrying out PCR amplification by using the specific primer provided by the invention; (3) and carrying out agarose gel electrophoresis experiment on the PCR product, and observing whether a specific band appears at the target position. The specific primer and the detection method for detecting trypanosoma protozoa provided by the invention can quickly and accurately detect whether a target sample contains the trypanosoma protozoa, have high intra-genus universality and strong inter-genus specificity, and can be specially used for effectively detecting the trypanosoma protozoa. The invention also provides application of the primer.
Description
Technical Field
The invention relates to the field of molecular biology and pathogenic biology, in particular to a specific primer designed by using a partial sequence of a 60S RPL44 gene of Trypanosoma brucei, a detection method and application.
Background
Trypanosoma (Trypanosoma sp.) protozoa are now about twenty-few, and are commonly parasitic in fish, amphibians, reptiles, birds, mammals, and human blood or tissue cells. Trypanosomes parasitizing humans can be divided into two broad categories, i.e., salivary-derived trypanosomes transmitted through saliva and fecal-derived trypanosomes transmitted through feces, depending on the route of infection. Trypanosomes having severe pathogenicity to livestock include trypanosoma brucei (t.brucei), trypanosoma congolense (t.congolense), trypanosoma evansi (t.evansi), etc., and species having severe pathogenetic effects to human include trypanosoma brucei and trypanosoma cruzi (t.cruzi). Trypanosomiasis (trypanosomisis) is one of the key prevention and treatment objects listed by WHO. Trypanosoma brucei parasitizing livestock and Trypanosoma leyi parasitizing mice are commonly used research materials in laboratories. Although our country is not a human trypanosomiasis epidemic area, the risk of the input trypanosomiasis is greatly increased with the increasing frequency of international communication. In 8 months in 2014, 1 case of input trypanosomiasis exists in Shanghai Changhai hospitals, and two cases are increased in 2017, so that an effective method for diagnosing trypanosomes is very necessary to be established as a technical reserve.
The prior art reports a method for detecting trypanosoma protozoa by adopting a PCR technology, but the following defects still exist: 1) the existing method has low universality aiming at the detection of trypanosoma protozoa, can only detect one or two of the trypanosoma protozoa to be positive, and can not detect other trypanosoma protozoa; 2) the existing method still has a defect of lack of specificity for detecting trypanosoma protozoa, and the detection result is influenced by other blood-borne protozoa.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a group of specific primers for detecting trypanosoma protozoa, which can detect various trypanosoma protozoa and have the characteristic of strong intra-genus universality.
In order to solve the technical problems, the invention provides a group of specific primers for detecting trypanosoma protozoa, and the nucleotide sequences of the specific primers are as follows:
upstream primer P1: 5' -CCTGATGAAAGGTGCAATG (shown as SEQ ID NO:1) or a complementary sequence thereof;
the downstream primer P2: 5' -CGTTTTCGCCTTCTTGTGGA (shown in SEQ ID NO:2) or a complementary sequence thereof.
Preferably, the upstream primer is shown as SEQ ID NO. 1, and the downstream primer is shown as SEQ ID NO. 2.
The specific primers provided by the invention are shown as SEQ ID NO. 1 (or a complementary sequence thereof) and SEQ ID NO. 2 (or a complementary sequence thereof), have high universality when used for detecting trypanosoma protozoa, and can be used for detecting at least 8 trypanosoma protozoa including trypanosoma cruzi (XM _806327.1), trypanosoma brucei rhodesiense (XM _011773651.1), trypanosoma congolense (TcIL3000.11.3000), trypanosoma evansi (TevsTIB805.2.3460), active trypanosoma (T.vivax) (TvY486_1103320), trypanosoma donii (T.randelii) (TRSC58_05400) and trypanosoma griseki (T.grayii) (DQ04_ 03581020).
The specific primers are shown in SEQ ID NO. 1 and SEQ ID NO. 2, have strong specificity when used for detecting trypanosoma protozoa, and have negative detection results on the following 24 other genera of hematogenous protozoa which are common in public databases and hematoparasite examination. The 24 other genera of blood-borne protozoa include: babesia microti (genbank ID: XM _012792916.1), babesia gemmifera (b.bigemina) (EuPathDB ID: BBBOND _0200890), plasmodium vivax (XM _001613016.1), plasmodium falciparum (XM _001351084.1), plasmodium malariae (p.malariae) (PmUG01_08051800), plasmodium ovale subspecies (p.ovale cutisi) (PocGH01_08043600), plasmodium falciparum (p.reiculatum) (PRELSG _0831300), plasmodium galamrini (p.gabonii) (PGSY 8_ 6866), plasmodium lismortis (p.reicherhei) (PRCDC 0303700), plasmodium gallinarum (p.gallinarallinaceum) (PGAL 8_ A _00003700), plasmodium gondii (XM _002367604.2), leishmania donovani (l _003864113.1), plasmodium falciparum (p.galbana) (l), plasmodium falciparum strain (l) (lbr _ 4633.p.7), plasmodium falciparum strain (l) (lbr _ 4633.p.4633), plasmodium falciparum l (l) (lmr l) Alexabica (L.arabica) (LARLEM1108_130021200), L.gerbili (LGELEM452_330043100), L.turancis (L.turanica) (LTULEM423_330043200), L.mexicana (Lmexicana) (LmxM.32.3230), L.aethiopica (LAEL147_000189300), and L.endermis (L.encietti) (LENLEM3045_ 330042300).
The group of specific primers provided by the invention is shown as SEQ ID NO. 1 and SEQ ID NO. 2, has strong specificity for the detection of trypanosoma protozoa, can specifically detect a plurality of protozoa in the trypanosoma, and has negative detection results for blood-borne protozoa of other genera.
The invention also provides a method for specifically detecting trypanosoma protozoa, which comprises the following steps:
(1) extracting the genomic DNA of a target sample;
(2) taking the genomic DNA of the target sample extracted in the step (1) as a template, and carrying out PCR amplification by using specific primers shown in SEQ ID NO:1 (or a complementary sequence thereof) and SEQ ID NO:2 (or a complementary sequence thereof);
(3) and carrying out agarose gel electrophoresis experiment on the PCR product, and observing whether a specific band appears at the target position.
Preferably, the specific primer is a sequence shown as SEQ ID NO. 1 and SEQ ID NO. 2.
In particular toThe PCR reaction system in the step (2) comprises: 6 to 18 mu l H2O, 1-3 mul 10 Xbuffer (TaKaRa), 1-3 mul dNTP, 0.2-0.6 mul Taq, 0.5-1.5 mul of specific upstream and downstream primers respectively, and 1-3 mul genomic DNA of a target sample.
Preferably, the reaction system is: the total volume of each PCR reaction was 20. mu.l, containing 11.6. mu. l H2O, 2. mu.l of 10 XBuffer (TaKaRa), 2. mu.l of dNTP, 0.4. mu.l of Taq, 1. mu.l of each of specific upstream and downstream primers, and 2. mu.l of genomic DNA of the target sample.
Specifically, the PCR amplification procedure in the step (2) is as follows:
firstly, pre-denaturation is carried out for 5min at 94 ℃;
② denaturation at 94 ℃ for 30 s;
③ annealing at 56 ℃ for 30 s;
extension at 72 ℃ for 20 s;
fifthly, repeating the steps from the step two to the step four for 30 to 40 cycles, and extending for 5 to 15min at the temperature of 72 ℃.
Preferably, the steps from (c) - (d) are repeated for 35 cycles and then extended for another 10min at 72 ℃.
Specifically, in the step (3), after the electrophoresis is finished, the agarose gel is placed under a gel imager for observation, and whether a specific band appears at the 156bp position of the target position, that is, whether the target sample contains trypanosoma protozoan DNA is checked.
The invention also provides application of specific primers shown as SEQ ID NO. 1 (or a complementary sequence thereof) and SEQ ID NO. 2 (or a complementary sequence thereof) in detecting whether a target sample contains trypanosoma protozoa. The applications include use in research, teaching, commercial services and other activities relating to the fields of molecular biology, pathogenic biology and the like.
The invention also provides application of specific primers shown as SEQ ID NO. 1 (or a complementary sequence thereof) and SEQ ID NO. 2 (or a complementary sequence thereof) in preparing a detection tool for detecting trypanosoma protozoa. The detection tool is a common detection tool in the field of biological detection, and comprises a gene chip, a detection test paper or a detection kit and the like.
The invention also provides a detection tool for preparing trypanosoma protozoa, which comprises specific primers shown as SEQ ID NO. 1 (or a complementary sequence thereof) and SEQ ID NO. 2 (or a complementary sequence thereof). Preferably, the specific primer is a sequence shown as SEQ ID NO. 1 and SEQ ID NO. 2.
Specifically, the detection tool is a common detection tool in the field of biological detection, and comprises a gene chip, detection test paper or a detection kit and the like.
Specifically, the detection tool is a PCR detection kit, the PCR detection kit comprises various reagents for completing PCR, wherein specific primers shown as SEQ ID NO:1 and SEQ ID NO:2 are contained, and the specific primers respectively comprise 0.5-1.5 mu l, and in addition, the detection tool further comprises: 6 to 18 mu l H2O、1～3μl 10×Buffer(TaKaRa)、1～3μl dNTP、0.2～0.6μl Taq。
Preferably, the PCR detection kit comprises 1 μ l of each specific primer shown as SEQ ID NO. 1 and SEQ ID NO. 2, and further comprises: 11.6 μ l H2O、2μl 10×Buffer(TaKaRa)、2μl dNTP、0.4μl Taq。
The specific primers shown as SEQ ID NO. 1 (or a complementary sequence thereof) and SEQ ID NO. 2 (or a complementary sequence thereof) provided by the invention have strong specificity when used for detecting trypanosoma protozoa; the kit can detect a plurality of protozoa (at least 8 kinds) in the trypanosoma, and has high intra-genus universality; the detection results of common hematogenous protozoa of other genera in a public database and in hematoparasite examination are negative, and the specificity is very strong; the method is beneficial to quickly screening the non-trypanosoma protozoan infected sample during trypanosoma protozoan detection, improves the efficiency of epidemic prevention inspection, and can recommend further detection aiming at the trypanosoma protozoan infected sample.
The method for specifically detecting trypanosoma protozoa provided by the invention can quickly and accurately detect whether the target sample contains the trypanosoma protozoa, has high intra-genus universality and strong inter-genus specificity, and can be specially used for effectively detecting the trypanosoma protozoa.
Drawings
FIG. 1 is a diagram showing the alignment of the 60S RPL44 gene sequence of Trypanosoma brucei with the homologous sequences of 24 other genera of blood-borne protozoa in the public database.
FIG. 2 is a schematic representation of the alignment of the 60S RPL44 gene sequence of Trypanosoma brucei with homologous sequences from other Trypanosomes within 8 species in the public database.
FIG. 3 is a schematic diagram showing the results of detection of a trypanosoma protozoan sample in example 1, in which M is a DNA marker; 1-3 are the template DNA of trypanosoma brucei, trypanosoma brucei rhodesiense and trypanosoma congolense respectively; 4-8 represent five concentration gradients 10 ng/. mu.l, 1 ng/. mu.l, 100 pg/. mu.l, 10 pg/. mu.l, 1 pg/. mu.l of trypanosoma evansi template DNA, 9 is a blank control.
FIG. 4 is a diagram showing the results of detection of other genus blood-derived protozoa in example 2, in which all of the protozoan template DNAs were used at a concentration of 1 ng/. mu.l, and M is a DNA marker; 1 is a Babesia microti template DNA; 2 is Leishmania donovani template DNA; 3 is plasmodium vivax template DNA; 4 is plasmodium falciparum template DNA; 5 is Toxoplasma gondii template DNA; 6 is trypanosoma evansi template DNA; 7 is negative mouse serum DNA; and 8 is negative human serum DNA.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The pair of specific primers SEQ ID NO:1 and SEQ ID NO:2 specially used for detecting trypanosoma is obtained by comparing a gene sequence (comprising a 60S RPL44 gene) of the trypanosoma brucei with homologous sequences of 24 other genera of blood-derived protozoa (comprising the Paecilomyces microti, Plasmodium vivax, Plasmodium falciparum, Toxoplasma gondii, Leishmania donovani and the like) in a public database and other trypanosomes (comprising the Trypanosoma brucei rhodesiense, Trypanosoma congolense, trypanosoma evansi and the like) in 8 species, and simultaneously combining the scoring condition of a plurality of pairs of primers designed by the Primer Premier 6 on the gene sequence (comprising the SRPL44) of the Trypanosoma brucei, and verifying and comprehensively screening by a plurality of detection tests. The length of the target fragment amplified by the primer is 156 bp.
The light grey parts in fig. 1 and 2 are highly specific fragments of trypanosoma screened by the inventors and corresponding fragments of other protozoa, the length is 156bp, the dark grey parts are corresponding primer parts screened by the inventors, and the bases of the sequence are the same.
The inventor finds through experiments that specific primers SEQ ID NO:1 and SEQ ID NO:2 designed and synthesized aiming at the 60S RPL44 gene sequence of the trypanosoma brucei subsp have very remarkable intra-genus universality and intergeneric specificity when used for detecting trypanosoma protozoa, can detect obvious specific bands for most species (at least 8 species related to the invention) of the trypanosoma protozoa, and have negative detection results for blood-derived protozoa (at least 24 species related to the invention) of other species common in hematoparasite examination.
Example 1 intra-genus versatility of specific primers of the invention specifically detecting trypanosoma protozoa:
the following method is adopted for detection:
(1) extracting the genomic DNA of a target sample;
DNA extraction was performed using DNeasy Blood & Tissue Kit (QIAGEN) according to the protocol. In addition, the method can also detect Whatman Classic FTA cards (WB120205) containing polypide, and the specific processing steps are as follows: a round piece with the diameter of 1.2mm is punched from a target area on an FTA card by a puncher (the hole diameter is 1.2mm, produced by Whatman company in UK), the round piece is placed in a PCR tube, the round piece is washed for 15min for 2-3 times by water, then the round piece is placed in an oven with the temperature of 80 ℃ for 10min, and then a PCR reaction system is added into the PCR tube, so that the DNA amplification can be realized.
The target samples include trypanosoma brucei (t.b.brucei) samples, trypanosoma brucei rhodesiense (t.b.rhodesiense) samples, trypanosoma congolense (t.congolense) samples, trypanosoma evansi (t.evansi) samples. Wherein the Trypanosoma brucei, Trypanosoma brucei rhodesiense and Trypanosoma congolense are provided by the worship subject group of the institute of Life sciences of the university of counterdenier, and the above 3 protozoa are all preserved in Whatman Classic FTA card (WB 120205); the DNA of trypanosoma evansi is a DNA solution extracted by QIAGEN and provided by the Huangveyi subject group of animal science and technology college of Guangxi university.
(2) Taking the genomic DNA of each target sample extracted in the step (1) as a template, and carrying out PCR amplification by using the primers SEQ ID NO. 1 and SEQ ID NO. 2 provided by the invention;
the reaction system is as follows: the total volume of each PCR reaction was 20. mu.l, containing 11.6. mu. l H2O, 2. mu.l of 10 XBuffer (TaKaRa), 2. mu.l of dNTP, 0.4. mu.l of Taq, 1. mu.l of each of specific upstream and downstream primers, and 2. mu.l of genomic DNA of a target sample;
the PCR amplification procedure was: (1) pre-denaturation at 94 ℃ for 5 min; (2) denaturation at 94 ℃ for 30 s; (3) annealing at 56 ℃ for 30 s; (4) extension at 72 ℃ for 20 s; (5) repeating the steps (2) - (4) for 35 cycles, and extending for 10min at 72 ℃.
(3) And carrying out agarose gel electrophoresis experiment on the PCR product, observing the run agarose gel under a gel imager, and checking whether a specific band appears at the target position.
The detection results show that in the test, the trypanosoma brucei (t.b.brucei) sample, the trypanosoma brucei rhodesiense (t.b.rhodesiense) sample, the trypanosoma congolense (t.congolense) sample and the trypanosoma evansi (t.evansi) sample can detect a very obvious specific band at the expected target of 156bp, the positive detection rate is 100%, and the detection results of the 4 samples are shown in a schematic diagram in fig. 3. Through verification, the other four trypanosoma protozoa related to the invention also show consistent detection results.
Because more samples are difficult to obtain, the invention does not carry out example detection on more trypanosomes, but the pair of primers can theoretically detect other trypanosomes in the genus through homologous sequence comparison (the part of the primers have high sequence homology, and the 3' end bases of the upstream primer and the downstream primer are the same).
Example 2 intergeneric specificity of specific primers of the invention for the specific detection of trypanosoma protozoa:
the following method is adopted for detection:
(1) extracting the genomic DNA of a target sample;
DNA extraction was performed using DNeasy Blood & Tissue Kit (QIAGEN) according to the protocol.
The target samples including the following 5 species of hematogenous protozoa commonly found in the examination of hematoparasites were all negative. The 5 other genera of blood-borne protozoa include: babesia microti (Babesia microti), Leishmania donovani (Leishmania donovani), Plasmodium vivax (Plasmodium vivax), Plasmodium falciparum (Plasmodium falciparum), and Toxoplasma gondii (Toxoplasma gondii) are provided by the central for the prevention and control of parasites in the chinese center for the prevention and Control of Disease (CDC).
(2) Taking the genomic DNA of each target sample extracted in the step (1) as a template, and carrying out PCR amplification by using the primers SEQID NO. 1 and SEQ ID NO. 2 provided by the invention;
the reaction system is as follows: the total volume of each PCR reaction was 20. mu.l, containing 11.6. mu. l H2O, 2. mu.l of 10 XBuffer (TaKaRa), 2. mu.l of dNTP, 0.4. mu.l of Taq, 1. mu.l of each of specific upstream and downstream primers, and 2. mu.l of genomic DNA of a target sample;
the PCR amplification procedure was: (1) pre-denaturation at 94 ℃ for 5 min; (2) denaturation at 94 ℃ for 30 s; (3) annealing at 56 ℃ for 30 s; (4) extension at 72 ℃ for 20 s; (5) repeating the steps (2) - (4) for 35 cycles, and extending for 10min at 72 ℃.
(3) And carrying out agarose gel electrophoresis experiment on the PCR product, observing the run agarose gel under a gel imager, and checking whether a specific band appears at the target position.
The detection result shows that no specific band is detected at the expected target 156bp position by each blood-borne protozoon in the test, and the detection results are all negative. Although no more other blood-borne protozoa are detected by way of example, the pair of primers is found to be incapable of detecting other genus protozoa by homologous sequence alignment (the primers have low partial sequence homology and the 3' end bases of the upstream and downstream primers are greatly different).
In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> university of Compound Dan
<120> specific primer for detecting trypanosoma protozoa, detection method and application
<130> CPC-NP-17-100821
<160> 2
<170> PatentIn version 3.4
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<223> primer
<400> 1
CCTGATGAAA GGTGCAATG 19
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<223> primer
<400> 2
CGTTTTCGCC TTCTTGTGGA 20
Claims (6)
1. A method for non-disease diagnostic purposes for the specific detection of trypanosoma protozoa, comprising the steps of:
(1) extracting the genomic DNA of a target sample;
(2) taking the genomic DNA of the target sample extracted in the step (1) as a template, and performing PCR amplification by using a specific primer; the sequence of the specific primer is as follows:
the upstream primer is as follows: the sequence shown as SEQ ID NO. 1;
the downstream primer is: the sequence shown as SEQ ID NO. 2;
(3) and carrying out agarose gel electrophoresis experiment on the PCR product, and observing whether a specific band appears at the position of 156bp of the target position.
2. The method of claim 1, wherein the PCR reaction system in step (2) comprises: 6 to 18 mu l H2O, 1-3 mul 10 Xbuffer, 1-3 mul dNTP, 0.2-0.6 mul Taq, 0.5-1.5 mul of specific upstream and downstream primers respectively, and 1-3 mul genomic DNA of a target sample.
3. The method of claim 2, wherein the PCR reaction system is: the total volume of each PCR reaction was 20 μ l, containing 11.6 μ l H2O, 2. mu.l of 10 XBuffer, 2. mu.l of dNTP, 0.4. mu.l of Taq, 1. mu.l of each of specific upstream and downstream primers, and 2. mu.l of genomic DNA of the target sample.
4. The method of claim 1, wherein the PCR amplification procedure in step (2) is:
firstly, pre-denaturation is carried out for 5min at 94 ℃;
② denaturation at 94 ℃ for 30 s;
③ annealing at 56 ℃ for 30 s;
extension at 72 ℃ for 20 s;
and step two to step four, after 30 to 40 cycles of repetition, the extension is carried out for 5 to 15min at 72 ℃.
5. The method of claim 4 wherein steps (c) - (d) are repeated for 35 cycles and then extended for a further 10min at 72 ℃.
6. The application of a specific primer in preparing a kit for detecting trypanosoma protozoa is characterized in that the sequence of the specific primer is as follows:
the upstream primer is as follows: the sequence shown as SEQ ID NO. 1;
the downstream primer is: the sequence shown as SEQ ID NO. 2.
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| CN103911447A (en) * | 2014-04-03 | 2014-07-09 | 河北国际旅行卫生保健中心 | Primers, probes and method for detecting plasmodium |
| WO2015170324A3 (en) * | 2014-05-04 | 2016-03-10 | Forrest Innovations Ltd. | Compositions for mosquito control and uses of same |
| CN107326024A (en) * | 2017-09-07 | 2017-11-07 | 辽宁省农业科学院大连生物技术研究所 | A kind of high flux rapid extraction plasmodium DNA method and its application |
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