CN113293223B - Application of primer combination in preparation of reagent for detecting bovine babesia ovale and anaplasma by adopting double PCR (polymerase chain reaction) method - Google Patents
Application of primer combination in preparation of reagent for detecting bovine babesia ovale and anaplasma by adopting double PCR (polymerase chain reaction) method Download PDFInfo
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Abstract
The invention relates to application of a primer combination in preparing a reagent for detecting bovine babesia ovale and anaplasma by a double PCR (polymerase chain reaction) method. The method comprises the steps of designing and synthesizing 2 pairs of specific primers P1 and P2 according to a bovine oval babesia AMA1 gene (KT312793) conserved sequence and a bovine anaplasma 16S rRNA gene conserved sequence, adding an upstream primer and a downstream primer of a specific primer sequence P1 and an upstream primer and a downstream primer of a specific primer sequence P2 and a PCR mixed solution into a PCR reaction tube, mixing, and adding bovine oval babesia DNA and bovine anaplasma DNA extracted from a blood sample to be detected; placing the PCR tube in a PCR amplification instrument for circulation; the PCR product was electrophoresed on a 1.5% agarose gel. The results show that the invention has the characteristics of rapidness, specificity, sensitivity, high efficiency, low cost and the like, and is convenient for clinical application.
Description
Technical Field
The invention belongs to the technical field of molecular diagnosis, and particularly relates to application of a primer combination in preparation of a reagent for detecting bovine babesia ovale and anaplasma by a double PCR (polymerase chain reaction) method.
Background
Bovine Babesia ovocystis disease (Babesia ovata) is a parasitic disease caused by the parasitism of Babesia species within bovine red blood cells. Many cattle with illness are characterized by high fever, anemia, jaundice, and hemoglobinuria, which often lead to death. At present, the diagnosis of the bovine ovoid babesia is mainly carried out through a blood smear and microscopic examination, the blood insect-staining rate of recessively infected cows is low, and missed detection and misdiagnosis are easy to occur in microscopic detection. Therefore, it is urgently needed to establish a diagnosis reagent with high sensitivity and strong specificity for early diagnosis of the disease.
Anaplasia (anaplasia) is a bacterial disease caused by anaplasia of the order Rickettsiales (Rickettsiales), anaplasia family (anaplasia), anaplasia genus (anaplama). The global distribution of anaplasma-free diseases is one of the important causes of huge economic losses in animal husbandry and threatening public health. Anaplasma bovis is the most common infectious agent, and clinical symptoms causing the disease are mainly characterized by body temperature rise, weight reduction, weakness, pale mucous membrane, lymphadenitis and even death.
Bovine anaplasma is a mixed infection with Babesia ovata and other blood pathogens, and its clinical symptoms appear similarly. Therefore, clinical symptoms and the defect of missed diagnosis or misdiagnosis easily occur in the blood smear diagnosis technology, and the advanced molecular diagnosis technology such as PCR detection has higher specificity and sensitivity, thereby leading the diagnosis of pathogens to be more reliable. The double PCR method can specifically detect 2 pathogens in one PCR amplification, and is more efficient and time-saving. Therefore, the invention provides the application of the primer combination in preparing the reagent for detecting the bovine babesia ovale and anaplasma by adopting a double PCR method.
Disclosure of Invention
The invention aims to provide application of a primer combination in preparing a reagent for detecting bovine babesia ovale and anaplasma by a double PCR method.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides application of a primer combination in preparing a reagent for detecting bovine Babesia ovata and anaplasma by a double PCR method, wherein the primers comprise 2 pairs of specific primers P1 and P2, wherein:
p1: the upstream primer is 5-GATACGGCTGTCGGTAGCAA-3 ', and the downstream primer is 5-GAGCTGTCACCATTGTCCTTAA-3';
p2: an upstream primer 5'-GGACTACGGTCGCAAGACTAAA-3'; the downstream primer is 5'-TCCACGATTACTAGCGATTCC-3'.
In the above technical solution, the reagent comprises a PCR amplification system, and the PCR amplification system comprises: taq PreMix, primer P1, primer P2, bovine genomic DNA template, and double distilled water (dd H)2O)。
In the technical scheme, the PCR amplification system is 25 mu L, wherein the Taq PreMix is 12.5 mu L, the primer P1 and the primer P2 are respectively 0.5 mu L, the DNA is 2 mu L, and dd H2O 9.5μL。
In the above technical scheme, the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 53-60 deg.C for 30s, 30 cycles, and final extension at 72 deg.C for 7 min.
In the above technical solution, the preferred annealing temperature is 56 ℃.
The invention has the beneficial effects that:
the primer combination is applied to the preparation of a reagent for detecting the babesia bovis and anaplasma by adopting a double PCR method, 2 pairs of specific primers P1 and P2 are designed and synthesized according to a conserved sequence of an AMA1 gene (KT312793) of the babesia bovis and an rRNA gene conserved sequence of the anaplasma bovis, an upstream primer and a downstream primer of a specific primer sequence P1 and an upstream primer and a downstream primer of a specific primer sequence P2 and a PCR mixed solution are added into a PCR reaction tube, and the extracted DN A of the babesia bovis and the anaplasma DNA of the blood sample to be detected are added after mixing; placing the PCR tube in a PCR amplification instrument for circulation; the PCR product was electrophoresed on a 1.5% agarose gel. The results show that the invention has the characteristics of rapidness, specificity, sensitivity, high efficiency, low cost and the like, and is convenient for clinical application.
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The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a graph showing the results of double PCR amplification, in which M: DL2000 DNA Marker; 1: an oval babesia DNA sample; 2: blank control.
FIG. 2 is a graph showing the results of a screening test for double PCR annealing temperatures, wherein M is DL2000 DNA Marker; 1: at 54 ℃; 2: 55 ℃; 3: 56 ℃; 4: 57 ℃; 5: at 58 ℃; 6: 59 ℃; 7: at 60 ℃.
FIG. 3 is a graph of the results of a specific amplification assay, wherein M: DL2000 DNA Marker; 1: babesia ovalifolia mixed with plasmaless DNA; 2: babesia bovis; 3: the sheep has no body; 4: babesia gemmifera; 5: theileria sergenti.
FIG. 4 is a graph showing the results of sensitivity tests, wherein M: DL2000bp DNA Marker; 1-8: the concentrations were 1.6 ng/. mu.L, 160 pg/. mu.L, 16 pg/. mu.L, 1.6 pg/. mu.L, 160 fg/. mu.L, 16 fg/. mu.L, 1.6 fg/. mu.L, 0.16 fg/. mu.L, respectively.
Detailed Description
The invention idea of the invention is as follows: the babesia ovalifolia is the most important insect species of bovine piroplasmosis, which seriously restricts the healthy development of the cattle raising industry and urgently needs to establish a quick detection method. The cattle Babesia ovata and anaplasma are transmitted by hard ticks, so that two pathogens of Babesia ovata and anaplasma mixed infection of cattle are common. Renneker et al found that the infection rate of anaplasma, Theileria and Babesia mixed in 195 sheep blood samples was 23%. Currently, methods for detecting Babesia ovalis and anaplasma include blood smear detection, molecular biology detection, and serology detection. But the detection by blood smear is difficult to be distinguished and diagnosed from other blood pathogens, and the plasma-free amount in blood is small in the early stage of infection and is difficult to be detected by blood smear. Serological tests have high requirements on antigens and antibodies and can be established by screening various conditions. In molecular biology detection, although fluorescence quantitative PCR has high sensitivity, specificity and accuracy and can be used for quantitative analysis, the method has the defect of expensive reagents and experimental instruments, and the common PCR method is widely applied to epidemiological analysis due to the advantages of simple operation, high sensitivity and strong specificity.
The double PCR technology realizes the specific amplification of a plurality of target genes in the same system, and the design of primers is very important. The double PCR detection method established by the invention selects the Babesia ovata A MA1 gene on the target gene, and AMA1 is protein secreted to the surface of the polypide in a membrane binding mode before the polypide invades erythrocytes, so that the double PCR detection method has higher conservation. The plasma-free method selects the conserved gene 16S rRNA gene, the 2 gene sequences are just the more conserved sequences of the 2 pathogen-related genes, the self-specificity combination is not generated, no non-specificity amplification occurs except the target band in single PCR amplification or double PCR amplification, no cross reaction is seen, the specificity sensitivity is ensured, meanwhile, the 2 pathogens can be detected in the same time, and the detection time is greatly saved.
According to the invention, a gene conserved sequence target gene locus is found according to an oval babesia AMA1 gene sequence, a specific primer sequence P1 is designed, a gene conserved sequence is found as the target gene locus according to a bovine anaplasma 16S rRNA gene sequence, and a specific primer sequence P2 is designed; therefore, the invention designs and synthesizes 2 pairs of specific primers P1 and P2, wherein: p1: the upstream primer is 5-GATACGGCTGTCGGTAGCAA-3 ', and the downstream primer is 5-GAGCTGTCACCATTGTCCTTAA-3'; p2: an upstream primer 5'-GGACTACGGTCGCAAGACTAAA-3'; the downstream primer is 5'-TCCACGATTACTAGCGATTCC-3'. Based on the application of the primer combination in preparing the reagent for detecting the Babesia ovata and anaplasma bovis by adopting a double PCR method, the upstream primer and the downstream primer of the specific primer sequence P1 and the upstream primer and the downstream primer of the specific primer sequence P2 and a PCR mixed solution are added into a PCR reaction tube, and the DNA of the Babesia ovata and the anaplasma bovis extracted from a blood sample to be detected are added after mixing; placing the PCR tube in a PCR amplification instrument for circulation; the PCR product was electrophoresed on a 1.5% agarose gel. The invention detects the whole blood sample of suspected cattle cases in spring area, and compares the whole blood sample with blood smear microscopy, routine PCR and fluorescence quantitative PC R. The invention can specifically amplify the bands of bovine ovoid babesia and bovine anaplasma, and the sizes of the fragments are 1042bp and 475bp respectively. The method has no cross reaction with theileria sergenti, anaplasma ovis, and Babesia bovis and Babesia gemmifera. The lowest detection concentrations of the invention for bovine ovoid babesia and anaplasma are 16 fg/. mu.L and 1 fg/. mu.L. The double PCR detection is carried out on 30 collected blood samples, and the bovine ovobabesia positive rate is 30 percent (9/30) and the bovine anaplasma positive rate is 46.67 percent (14/30). The invention has higher specificity and sensitivity, and can be used for the rapid diagnosis and epidemiological investigation of bovine Babesia ovata and bovine anaplasma.
Examples
1. Materials and methods
1.1 sources of pathological material
The blood samples were collected from 30 portions of Yanbian yellow cattle raised in Jilin province 29682and spring, anticoagulated blood was aseptically collected and washed three times with PBS. Stored at-20 ℃ for later use. Meanwhile, a blood smear is made and fixed and stored by formaldehyde for staining microscopy. The bovine babesi and geminibarbus insect standard genomic DNA was gifted by professor Xuansheng university of domestic animal and university in Japan. Theileria sergenti genomic DNA was stored by the preventive veterinary laboratory at the university of Yanbian.
1.2 major reagents
Whole blood DNA Extraction Kit, Taq DNA polymerase, dNTP, Agarose Gel DNA Extraction Kit, DL2000 DNA Marker, etc. were purchased from Bao bioengineering (Dalian) Co., Ltd.
1.3 application of primer combination in preparation of reagent for detecting bovine Babesia ovata and anaplasma by adopting double PCR method
1.3.1 design and Synthesis of primers 2 pairs of specific primers were designed based on the GenBank accession of the oval Babesia AMA1 gene sequence (KT312793), plasmaless 16S rRNA gene sequence (MH255941) and using Primer Premier 5.0 software. The upstream primer of P1 is 5-GATACGGCTGTCGGTAGCAA-3 ', and the downstream primer is 5-GAGCTGTCACCATTGTCCTTAA-3'. The size of the amplified target fragment is 1042 bp; p2 upstream primer 5'-GGACTACGGTCGCAAGACTAAA-3', downstream primer 5'-TCCACGATTACTAGCGATTCC-3', fragment size 475bp, was synthesized by Shanghai Yingjun Biotech.
1.3.2 preparation of bovine Whole blood DNA Standard template genomic DNA was extracted according to the procedure of the Whole blood genomic DNA extraction kit, the extracted DNA was dissolved in 50. mu.LTE and stored at-20 ℃ for further use.
1.3.3 double PCR amplification Using extracted bovine genomic DNA as template, the PCR amplification system was 25. mu.L: 12.5 mu L of Taq PreMix, 0.5 mu L of each upstream and downstream primer of AMA1 and 16S rRNA; 2 mu L of DNA; dd H2O9.5 mu L; the PCR reaction conditions were as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing-extension at 55 ℃ for 30s, 30 cycles, and final extension at 72 ℃ for 7 min.
1.3.4 double PCR annealing temperature screening with extracted bovine genome DNA as template, annealing temperature gradient of 53-60 deg.c and other amplification parameters as 1.3.3.
1.3.5 Duplex PCR specificity test Using optimized duplex PCR to respectively perform duplex PCR amplification on the genomic DNA of Babesia ovata, Bolus agaplasma, Babesia bovis, Babesia gemmifera and Theileria sergentii as templates, using double distilled water as a blank control, and analyzing the specificity by agarose gel electrophoresis.
1.3.6 sensitivity test the D of the extracted bovine genomic DNA was determined using an ultraviolet spectrophotometer260nmValue, calculate the DNA content. And (3) continuously diluting the mixture into 7 different concentrations by taking 10 times as a gradient, performing double PCR amplification according to the optimal optimized condition, and detecting the sensitivity of the method.
1.4 test of clinical specimens 30 bovine blood samples taken from \29682ofJilin province, spring were tested using the present invention.
2. Results
2.1 double PCR amplification adopts optimized PCR reaction conditions, PCR amplification is carried out by taking bovine genome DNA as a template, and then 10g/L agar gel electrophoresis detection shows that specificity bands of 1042bp and 475bp respectively appear (see figure 1), and the sizes of the bands are consistent with the expected fragment sizes.
2.2 the screening annealing-extension temperature gradient of the duplex PCR annealing temperature is 54 ℃ to 60 ℃. Among them, the target band was brightest at 56 ℃ and, therefore, 56 ℃ was set as the optimum annealing temperature (see FIG. 2).
2.3 Dual PCR specificity test Using bovine Babesia ovata and bovine anaplasma-free mixed infected DNA, bovine Babesia, Babesia gemmifera and Theileria sergenti genomic DNA as templates, PCR amplification was performed according to the optimal reaction conditions, and PCR products were analyzed by 10g/L agarose gel electrophoresis. The electrophoresis results showed that the expected band was amplified by mixed infection of bovine babesia ovaliformis and anaplasma-free, whereas the amplified bands did not appear in any of the genomic DNA of babesia bovis, ovine anaplasma, babesia gemmifera and theileria sergenti as control samples (see FIG. 3).
2.4 double PCR sensitivity test the anticoagulated blood of the mixed infected Bobei Babesia ova and Boniu plasmaless is extracted to DNA, the DNA concentration is determined by spectrophotometry, 10 times of gradient dilution is carried out in sequence, double PCR amplification is carried out respectively, and agarose gel electrophoresis detection is carried out on the reaction result (see figure 4). The lowest content of DNA of the bovine egg-shaped Babesia was detected to be 16 fg/. mu.L, and the lowest content of DNA of bovine anaplasma was detected to be 1 fg/. mu.L.
2.5 Dual PCR clinical sample detection
The double PCR detection of 30 blood samples collected from areas of \/29682and spring of Jilin province shows that the positive rate of the bovine Babesia ovata is 30% (18/30), the positive rate of the bovine anaplasma is 46.67% (14/30) and the mixed infection rate is 46.67% (14/30).
30 clinical samples are detected by the invention. The result shows that the method has better practical application effect, does not need to carry out fussy pathogen separation and other auxiliary tests, and has shorter time consumption. Compared with the traditional blood smear staining microscopic examination, the specificity and the sensitivity are greatly improved, the false positive rate of clinical samples is also greatly reduced, and the method is worthy of popularization and application in basic veterinary departments.
In conclusion, the invention can simultaneously detect the bovine ovoid babesia and the bovine anaplasma, has good specificity and repeatability, simple and convenient operation, time and labor saving, and can be used for rapid differential diagnosis of two pathogen infections and molecular epidemiological investigation.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
<110> Jilin agriculture science and technology institute
Application of <120> primer combination in preparation of reagent for detecting bovine ovoid babesia and anaplasma by using dual PCR method
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Claims (5)
1. Use of a primer combination for preparing a reagent for detecting Babesia ovata and anaplasma bovis by a double PCR method, wherein the primers comprise 2 pairs of specific primers P1 and P2, wherein:
p1: the upstream primer is 5'-GATACGAGGCTGTCGGTAGCAA-3', and the downstream primer is 5'-GAGCTGTCACCATTGTCCTTAA-3';
p2: an upstream primer 5'-GGACTACGGTCGCAAGACTAAA-3'; the downstream primer is 5'-TCCACGATTACTAGCGATTCC-3'.
2. Use of a primer combination according to claim 1 for the preparation of a reagent for the detection of bovine Babesia ovata and anaplasma using a duplex PCR method, wherein the reagent comprises a PCR amplification system comprising: taq Premix, primer P1, primer P2, bovine genomic DNA template and dd H2O。
3. Use of the primer combination according to claim 2 for preparing a reagent for detecting Babesia ovata and anaplasma bovis by using a dual PCR method, wherein the PCR amplification system is 25 μ L, wherein the Taq PreMix 12.5 μ L, the primer P1 and the primer P2 are 0.5 μ L each, the DNA is 2 μ L, and the dd H is 0 μ L2O 9.5μL。
4. Use of the primer combination according to claim 2 for preparing a reagent for detecting Babesia ovata and anaplasma bovis by using a dual PCR method, wherein the PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 53-60 deg.C, extension for 30s, 30 cycles, and final extension at 72 deg.C for 7 min.
5. Use of the primer combination according to claim 4 for preparing a reagent for detecting Babesia ovata and anaplasma bovis by the duplex PCR method, wherein the annealing temperature is 56 ℃.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088149A (en) * | 2013-02-28 | 2013-05-08 | 中国农业科学院兰州兽医研究所 | Kit for detecting anaplasma bovis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088149A (en) * | 2013-02-28 | 2013-05-08 | 中国农业科学院兰州兽医研究所 | Kit for detecting anaplasma bovis |
Non-Patent Citations (3)
| Title |
|---|
| Accession No. KT312793;Niu Q.等;《Genbank》;20151018;全文 * |
| Molecular characterization of South Indian field isolates of bovine Babesia spp. and Anaplasma spp.;Rangapura Kariyappa Pradeep等;《Parasitology Research》;20181217;第118卷(第2期);617-630 * |
| 多重PCR技术在寄生虫病诊断上的应用及其建立方法的浅析;石云良等;《广西畜牧兽医》;20101231;第26卷(第1期);59-60 * |
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