CN111593132B - Molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity - Google Patents
Molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity Download PDFInfo
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Abstract
The invention discloses a molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity, and relates to a molecular typing method for different strains of treponema pallidum, which is established by utilizing the TP0136 protein heterogeneity of treponema pallidum. The method is used for distinguishing different subtypes of treponema pallidum by detecting the heterogeneity of the TP0136 genes of different strains of treponema pallidum, so that the defects of a treponema pallidum molecular typing detection method are overcome; the method has the characteristics of definite detection result and high sensitivity, and is easy to popularize.
Description
Technical Field
The invention relates to a molecular typing method for different strains of treponema pallidum, in particular to a molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity for non-diagnostic or therapeutic purposes.
Background
Syphilis is a sexually transmitted disease caused by treponema pallidum infection, and is the third place of infectious disease in China. Congenital transmission of treponema pallidum is a leading factor in the death of fetuses and perinatal infants in developing countries. Early mucosal lesions (chancroid) caused by treponema pallidum infection provide entry of HIV into the portal, increasing the risk of infection and transmission of HIV, and pose a serious threat to public health. Therefore, the effective prevention and control of treponema pallidum infection is urgent. The treponema pallidum molecular typing system is not only an important means for developing treponema pallidum molecular epidemiological researches, is helpful for explaining the relationship between genotype and virulence and invasiveness of the strain, but also can evaluate the relationship between genotype and disease stage, disease outcome and clinical drug resistance, and provides basis for formulating treponema pallidum control strategies and effect evaluation thereof.
The American disease control center research team classifies the arp have into 21 subtypes of 2-22 according to the difference of the number of the repetitive fragments of the arp gene among treponema pallidum strains; according to RFLP map after tpr EGJ gene enzyme digestion, tpr is divided into 16 subtypes a-p, and a treponema pallidum molecular typing method, commonly called CDC typing, is established. Subsequently, marra et al used sequencing techniques to divide the TP0548 gene into a total of 9 subtypes a-i based on the heterogeneity of the TP0548 gene. Katz et al classified rpsA into 4 subtypes 8 to 11 based on the difference in the number of G repeats of rpsA gene. Since rpsA typing is not efficient, more research teams currently choose to combine CDC and TP0548 typing methods, known as new typing methods. In recent years, researchers have analyzed a plurality of variable loci in an attempt to select new loci such as TP0279, TP0558, TP0326, etc. for clinical differentiation, but typing efficiencies are not ideal. Therefore, the existing treponema pallidum typing method cannot meet clinical treponema pallidum diagnosis requirements, cannot distinguish treponema pallidum molecular types in nearly 90% of treponema pallidum infected people in China, cannot be accurately used for treponema pallidum molecular epidemiological research in China and clinical evaluation, and is in urgent clinical need for a more effective treponema pallidum molecular typing method.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: in order to overcome the defects in the prior art, a rapid, accurate, simple and convenient molecular typing method suitable for distinguishing different strains of treponema pallidum is provided, so as to facilitate differential diagnosis of treponema pallidum infection and molecular epidemiological research. We have found in previous studies that TP0136 has a genetic heterogeneity in different strains of treponema pallidum. By utilizing the characteristics, a novel treponema pallidum molecular typing method is established by researching TP0136 gene heterogeneity in different treponema pallidum strains. The invention utilizes the genetic heterogeneity of different strains TP0136 of treponema pallidum, establishes a new TP0136 molecular typing method based on molecular biology technology, and combines with the traditional three-gene typing method, and the established TP0136 molecular typing method is helpful for treponema pallidum molecular epidemiological research, reveals the association between treponema pallidum molecular typing and clinical characteristics, and is also helpful for further perfecting the traditional treponema pallidum molecular typing system.
In order to solve the technical problems, the invention adopts the following technical scheme:
a molecular typing method for distinguishing between different subtypes of treponema pallidum based on TP0136 gene heterogeneity for non-diagnostic or therapeutic purposes, said method being established using treponema pallidum TP0136 protein heterogeneity; the method comprises the following specific steps: passaging and extracting treponema pallidum strains; sequence analysis and primer design of treponema pallidum TP 0136; treponema pallidum TP0136DNA amplification, sequencing and typing.
Further, the treponema pallidum genus comprises 9 treponema pallidum and 23 clinical treponema pallidum isolates isolated from clinical patients with clinical treponema pallidum in Guangdong province; the 9 treponema pallidum strains comprise NicholsHouston strains, nichols Seattle strains, nichols Dallas strains, DAl-1 strains, bal73-1 strains, seattle81-4 strains, chicago strains, mexicoA strains and SS14 strains.
Further, the treponema strain passaging in step (1) is performed in New Zealand white rabbit testes.
Further, the 1X cell lysis buffer used in the extraction of the treponema pallidum strain described in step (1) comprises 10mM Tris,0.1M EDTA,0.5%SDS; the 2X cell lysis buffer contained 20mM Tris-HCl,0.2MEDTA,1%SDS.
Furthermore, the primer sequence used in the amplification of the treponema pallidum TP0136DNA in the step (3) is shown in SEQ ID NO. 1-3.
Further, the PCR amplification reaction reagent used in the step (3) for amplifying the treponema pallidum TP0136DNA comprises 5. Mu.L of the DNA sample to be detected, 200. Mu.M dNTPs, 5. Mu.L of 10X Go TaqPCR buffer, 1.5mM MgCl 2, 0.6. Mu.M TP0136 primer and 0.5U of hot start Taq PCR polymerase per 50. Mu.L of the PCR amplification reaction reagent.
Further, the condition of the treponema pallidum TP0136DNA amplification in the step (3) is controlled to be denatured at 95 ℃ for 10min;95 ℃ for 1min, 60 ℃ for 2min and 72 ℃ for 1min, 45 cycles in total; finally, the extension is carried out for 10min at 72 ℃.
Further, the treponema pallidum TP0136DNA sequencing in the step (3) is to carry out the sequencing by a 1.5% agarose gel electrophoresis identification and purification recovery of the PCR amplified product.
Compared with the prior art, the invention has the following beneficial effects:
The method is used for distinguishing different subtypes of treponema pallidum by detecting the heterogeneity of the TP0136 genes of different strains of treponema pallidum, so that the defects of a treponema pallidum molecular typing detection method are overcome; the method has the characteristics of definite detection result and high sensitivity, and is easy to popularize.
Drawings
FIG. 1 is an electrophoresis diagram of PCR products of treponema pallidum TP0136 gene;
Wherein, the left side is the syphilis TP0136 gene amplification product; the right side is the DNA molecular weight standard.
FIG. 2 is a graph showing the typing results of the treponema pallidum TP0136 gene;
Wherein, the three subtypes are classified into 6 subtypes according to the heterogeneity of the treponema pallidum TP0136 gene, wherein NicholsHouston, bal & lt 73 & gt-1 and Chicago have identical gene sequences and are classified into a first type; the gene sequences of Nichols Dallas and NicholsSeattle are identical and are classified as the second type; the remaining four forms are Dal-1, seattle 81-4, SS14 and Mexico A, respectively, in that order.
Detailed Description
A molecular typing method for distinguishing between different subtypes of treponema pallidum based on TP0136 gene heterogeneity for non-diagnostic or therapeutic purposes, said method being established using treponema pallidum TP0136 protein heterogeneity; the method comprises the following steps: passaging and extracting treponema pallidum strains; sequence analysis and primer design of treponema pallidum TP 0136; treponema pallidum TP0136 DNA amplification, sequencing and typing. The method comprises the following steps:
(one) New Zealand white rabbit testis passage and culture of treponema pallidum:
New Zealand white rabbit testes of 9 treponema pallidum are passaged and isolated: 2mL of frozen 9 standard strains of treponema pallidum (including Nichols Houston strain, nichols Seattle strain, nichols Dallas strain, DAl-1 strain, bal73-1 strain, seattle81-4 strain, chicago strain, mexicoA strain, and SS14 strain) with a strain of 1X 108 Tp/mL were inoculated into New Zealand white rabbit testes, and each testis was inoculated with 1 mL. 9 treponema pallidum strains proliferated in New Zealand white rabbit testes by means of New Zealand white rabbit intratestal inoculation. Prior to conducting the infection experiments, all New Zealand white rabbits were tested by venereal disease research Laboratory (VENEREAL DISEASE RESEARCH Laboratory, VDRL) and fluorescent treponema pallidum antibody adsorption (fluorescent treponemal antibody-absorbed, FTA-ABS) to exclude infection by rabbit treponema pallidum T.Paraluiscuiculi. Only VDRL and FTA-ABS negative animals were used for this experiment. The testes of the rabbits were checked daily for the occurrence of orchitis, and blood was drawn every three days to detect VDRL and FTA-ABS indicators in the serum. After the New Zealand white rabbits form orchitis and VDRL and FTA-ABS index changes positive in serum, the New Zealand white rabbits are euthanized, testes are cut off under aseptic operation, and the testes are cut up and centrifuged to prepare bacterial suspension of 1X 108 Tp/ml.
(II) extraction of treponema pallidum strain DNA:
9 extraction of treponema pallidum strain DNA: the spirochete extracted from infected rabbit testes was centrifuged at low speed, separated from rabbit tissue fragments, and then centrifuged using a microcentrifuge to precipitate treponema pallidum. Tp was resuspended in 1X lysis buffer (containing 10mM Tris,0.1M EDTA,0.5%SDS). Resuspension Tp was frozen at-20 ℃ or DNA was extracted using QIAAMP DNA MINI kit (QIAGEN) as needed. The method comprises the following specific steps: 1) Aspirate 20 μ LQIAGEN protease (or proteinase K) to the bottom of the 1.5mL centrifuge tube; 2) Adding 200 mu L of a sample of genome to be extracted into a centrifuge tube; 3. ) 200. Mu.L of buffer AL was added to the sample and vortexed for 15 seconds to mix. Complete mixing to obtain a homogeneous solution is important to ensure adequate lysis of the sample. If the sample volume is greater than 200. Mu.L, the amounts of protease and buffer AL are increased by equal amounts. In operation, care is taken not to add QIAGEN protease (or proteinase K) directly to buffer AL; 4) Incubation at 56 ℃ for 10 minutes, the DNA yield has reached a maximum under this condition, and extending the incubation time does not further increase the yield; 5) Rapidly centrifuging to remove droplets remaining in the 1.5mL centrifuge tube lid; 6) 200. Mu.L (equal volume of sample) of ethanol (96-100%) was added and mixed by vortexing for 15 seconds. After the vibration is finished, the solution is rapidly centrifuged to remove liquid drops remained in the cover of the centrifuge tube with the volume of 1.5 mL; 7, preparing a base material; the mixture from step 6 was transferred to QIAAMP MINI centrifuge columns (in a 2mL collection tube). The caps were snapped on (the caps of each centrifuge tube were closed during centrifugation to prevent aerosol generation during centrifugation), and 6000g was centrifuged for 1 minute. Placing QIAAMP MIN centrifugal column into a new clean 2mL receiving tube, discarding filtrate together with used collecting tube; 8) The QIAAMP MINI column was carefully opened and 500 μl buffer AW1 was added (note that the edge ring was not wetted). The lid was closed and centrifuged at 6000g for 1 min. The QIAAMP MINI cartridge was transferred to a new 2mL collection tube and the filtrate was discarded along with the used collection tube. Even if the amount of the initially added sample is more than 200. Mu.L, the amount of the buffer AW1 does not need to be increased in this step; 9) The filtrate was discarded, the QIAAMP MINI column was carefully opened and 500 μl of buffer AW2 was added (care was taken not to wet the limbal ring). The cover is covered tightly, and the maximum rotation speed is 20000g and the centrifugation is carried out for 3 minutes; 10 QIAAMP MINI centrifugation column was transferred to a new 2mL collection tube. Centrifugation is carried out for 1 minute at 20000g maximum speed. This step can help reduce the likelihood of buffer AW2 remaining; 11 QIAAMP MINI centrifugation column was transferred to a new 1.5mL collection tube. The column was carefully opened and 200 μl buffer AE or double distilled water was added (eluting volumes greater than 200 μl were not suitable for 1.5mL collection tubes, since the lower edge of the column would then come into contact with the eluate, resulting in the possibility of aerosol generation during centrifugation; eluting volumes less than 200 μl would significantly increase the concentration of DNA in the eluate but would decrease the overall DNA yield); For less than 1. Mu.g of DNA, 50. Mu.L of eluate is recommended; washing 2 times with 100. Mu.L of eluent was almost as effective as washing once with 100. Mu.L of eluent). Incubation for 1 min at room temperature (15-25 ℃) (generally, an increase in incubation time to 5 min increases the yield), followed by centrifugation at 6000g for 1 min. If the secondary elution is carried out with the addition of 200. Mu.L of buffer AE, the yield can be increased by about 15%. DNA was stored at-20 ℃. DNA was extracted for PCR detection and post-amplification procedures. DNA was stored at-20 ℃.
(III) Treponema TP0136 sequence analysis and primer design:
Sequence analysis of treponema pallidum TP 0136: TP0136 amino acid and nucleic acid sequences (http:// www.ncbi.nlm.nih.gov/genome) were searched from Genbank's protein and DNA databases and multiple sequence alignments were performed on 9 treponema pallidum (Nichols Houston, nichols Seattle, nichols Dallas, DAl-1, bal73-1, seattle81-4, chicago, mexicoA and SS 14) using BioEdit 7.1 software (http:// biokit.software.com/7.1 /).
Primer design for treponema TP 0136: primer design software was applied: THE PRIMER3: WWW primertool (http:// biotools. Umassmed. Edu/bioapps/Primer3_ www.cgi) primer design was performed on the gene sequence of the Nichols standard strain TP0136, which was queried in GenBank. The signal peptide of TP0136 was predicted using SignalP4.1Server software (http:// www.cbs.dtu.dk/services/SignalP /), and the open reading frame primers of TP0136 contained no signal peptide.
(IV) Treponema pallidum TP0136DNA amplification, sequencing and typing:
Treponema pallidum TP0136DNA amplification: conventional PCR amplified TP0136DNA of treponema pallidum 9 strains. The type 1 upstream primer is: 5'-CACAAGAGTCCGGGCCAGTG' (SEQ ID No: 1); the type 1 downstream primer is 5'-CAGCGGAGGGACCAGCAGCA' (SEQ ID No: 2), and the amplified product has a size of 196bp. The type2 and 3 upstream primers are 5'-CGCACGCCGCACGTCTATTT-3' (SEQ ID No: 3); the type2 and 3 downstream primers were: 5'-CAGCGGAGGGACCAGCAGCA-3' (SEQ ID No: 2), the amplified product sizes were 228 and 242bp, respectively. The type 4 upstream primer is 5'-CACAAGAGTCCGGACCAGTG-3' (SEQ ID No. 4); the type 4 downstream primer is: 5'-CAGCGGAGGGACCAGCAGCA-3' (SEQ ID No: 2), the amplification product sizes were 196bp, respectively. The type 5 and 6 upstream primers are 5'-CACAAGAGTCCGGACCAGTG-3' (SEQ ID No: 4); the type 5 and 6 downstream primers were: 5'-CAACGGAACGGCCGGCAGCA-3' (SEQ ID No: 5), the amplified products were 196bp in size. The 50. Mu.L PCR amplification reaction reagents contained: mu.L of DNA sample to be detected, 200 mu.M dNTPs, 5 mu.L of 10 xGo Taq PCR buffer, 1.5mM MgCl2, 0.6 mu.MTP 0751 primer, and 0.5U of hot start Taq PCR polymerase. Amplification conditions: denaturation at 95℃for 10min;95 ℃ for 1min, 60 ℃ for 2min and 72 ℃ for 1min, 45 cycles in total; finally, the extension is carried out for 10min at 72 ℃.
Treponema pallidum TP0136 amplification product sequencing and typing: amplified PCR products were identified by 1.5% agarose gel electrophoresis (see FIG. 1). And purifying and recycling the PCR amplified product by using an ExoSAP-IT PCR product purification kit. When the PCR amplification is complete, any unconsumed dNTPs and primers present in the PCR product mixture will affect the subsequent reaction. ExoSAP-IT uses two hydrolases, exoenzyme Exonuclease I and shrimp alkaline phosphatase Shrimp AlkalinePhosphatase, along with a specially formulated buffer to remove unwanted dNTPs and primers from the PCR product. Exonuclease I removes the remaining single stranded primer and any extraneous single stranded DNA that is produced in PCR. Shrimp alkaline phosphatase removes residual dNTPs from the PCR mixture, which can affect the subsequent reaction. ExoSAP-IT was added directly to the PCR product and then incubated at 37℃for 15 minutes. Enzymes are active in the buffer system of PCR and therefore do not require buffer exchange. After treatment, exoSAP-IT was inactivated by simply heating to 80 ℃ for 15 minutes. The use of ExoSAP-IT avoids all gel or column purification, precipitation, filtration, beads or magnetic separation. With ExoSAP-IT there was 100% yield of both short and long PCR products. The ORF of treponema pallidum TP0136 was sequenced using a two-way DNA sequencing method, and the sequencing results were subjected to multiple sequence alignment using BioEdit7.1 software to obtain typing results (see FIG. 2 in detail).
(V) typing capability verification of treponema TP 0136:
the method for typing the treponema pallidum TP0136 is applied to the typing of 23 clinical isolates of the treponema pallidum from dermatology hospitals of the university of south medical science. The results show that the types of 23 clinical isolates of treponema pallidum are 100% of treponema pallidum subspecies, suggesting high specificity of the method of the invention. Wherein 4 treponema pallidum clinical isolates (GD 003, GD008, GD022 and GD 023) were type I; the 16 treponema pallidum clinical isolates (GD 001, GD005, GD006, GD007, GD010, GD011, GD012, GD013, GD014, GD015, GD016, GD017, GD018, GD019, GD020, GD 021) were type 5; the 3 treponema pallidum clinical isolates (GD 002, GD004 and GD 009) were type 6.
Even if the Arp/Tpr/TP0548 three-genotyping method was used, only 23 clinical isolates of treponema pallidum could be classified as type 5 (13D/D, 14D/f, 14D/g, 15D/f, 16A/e); however, if the novel molecular typing method of Treponema TP0136 is combined with the traditional three-gene typing method, 23 clinical isolates in the study can be further divided into types 9 (13D/D/5, 13D/D/6, 14D/f/1, 14D/f/5, 14D/f/6, 14D/g/5, 15D/f/1, 15D/f/5 and 16A/e/5), and the typing sensitivity can be improved.
The invention has high specificity (100 percent), can be used for distinguishing spirochete infection of different spirochete genera, and is beneficial to differential diagnosis of clinical pathogens; and the sensitivity of the traditional genotyping can be improved, the genotyping efficiency is improved by 180%, and the clinical epidemiological level study is facilitated.
SEQUENCE LISTING
<110> Ke Wujian
<120> A molecular typing method for discriminating different subtypes of treponema pallidum based on TP0136 gene heterogeneity
<130> 2020
<160> 11
<170> PatentIn version 3.2
<210> 1
<211> 196
<212> DNA
<213> Artificial sequence
<400> 1
cacaagagtc cgggccagtg gggcgagtcg agccccacgc ccaaagcgag cgccgagcag 60
tatcggggca cggtcggtcg gtttgccgtg cagaaaatct acgtagttga aaaaaatggc 120
ggtgggaacg gtgtcgccgc gggtggggcg ggctgtcctg caaacgccag cagttccagc 180
ggagggacca gcagca 196
<210> 2
<211> 228
<212> DNA
<213> Artificial sequence
<400> 2
cgcacgccgc acgtctattt cccccagccc aacccccagc caacatctta tgggttgcgt 60
gcccgtccac gcccaaagcg agcgccgagc agtatcgggg cacggtcggt cggtttgccg 120
tgcagaaaat ctacgtagtt gaaaaaaatg gcggtgggaa cggtgtcgcc gcgggtgggg 180
cgggctgtcc tgcaaacgcc agcagttcca gcggagggac cagcagca 228
<210> 3
<211> 242
<212> DNA
<213> Artificial sequence
<400> 3
cgcacgccgc acgtctattt cccccagccc aacccccagc caacatctta tgggttgcgt 60
gcccgtccac gcccaaagcg agcgccgagc agtatcgggg cacggtcggt cggtttgccg 120
tgcagaaaat ctacgtagtt gaatctacgt agttgaaaaa aatggcggtg ggaacggtgt 180
cgccgcgggt ggggcgggct gtcctgcaaa cgccagcagt tccagcggag ggaccagcag 240
ca 242
<210> 4
<211> 196
<212> DNA
<213> Artificial sequence
<400> 4
cacaagagtc cggaccagtg gggcgagtcg agccccacgc ccaaagcgag cgccgagcag 60
tatcggggca cggtcggtcg gtttgccgtg cagaaaatct acgtagttga aaaaaatagc 120
ggtgggaacg gtgtcgccgc gggtggggcg ggctgtcctg caaacgccag cagttccagc 180
ggagggacca gcagca 196
<210> 5
<211> 196
<212> DNA
<213> Artificial sequence
<400> 5
cacaagagtc cggaccagtg ggacgagtcg agccccacgc ccaaagcgag cgccgagcag 60
tatcggggca cggtcggtcg gtttgccgtg cagaaaatct acgtagttga aaaaaatggc 120
ggtgggaacg gtgtcgccgc gggtggggcg ggctgtcctg caagcgccag cagtaccaac 180
ggaacggccg gcagca 196
<210> 6
<211> 196
<212> DNA
<213> Artificial sequence
<400> 6
cacaagagtc cggaccagtg ggacgagtcg agccccacgc ccaaagcgag cgccgagcag 60
tatcggggca cggtcggtcg gtttgccgtg cagaaaatct acgtagttga aaaaaatggc 120
ggtgggaacg gtgtcgccgc gggtggggcg ggctgtcctg caagcgccag cagtatcaac 180
ggaacggccg gcagca 196
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
cacaagagtc cgggccagtg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
cagcggaggg accagcagca 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
cgcacgccgc acgtctattt 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence
<400> 10
cacaagagtc cggaccagtg 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence
<400> 11
caacggaacg gccggcagca 20
Claims (1)
1. A molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity for non-diagnostic or therapeutic purposes, the method being established using treponema pallidum TP0136 protein heterogeneity, comprising the specific steps of:
step (1) passage of treponema pallidum strains and extraction of strain DNA;
Step (2) analyzing the sequence of the treponema pallidum TP0136 and designing a primer;
Step (3) treponema TP0136 DNA amplification, sequencing and typing;
The treponema pallidum comprises 9 treponema pallidum and 23 clinical treponema pallidum isolates separated from patients with clinical treponema pallidum in Guangdong province; the 9 treponema pallidum strains comprise Nichols Houston strain, nichols Seattle strain, nichols Dallas strain, DAl-1 strain, bal73-1 strain, seattle81-4 strain, chicago strain, mexicoA strain and SS14 strain;
The treponema pallidum strain passaging in step (1) is performed in testes of new zealand white rabbits;
The 1 Xcell lysis buffer used in the DNA extraction of the treponema pallidum strain described in step (1) comprises 10mM Tris,0.1M EDTA,0.5%SDS;
the primer sequence adopted in the step (3) of treponema pallidum TP0136DNA amplification is shown in SEQ ID NO. 1-5;
The PCR amplification reaction reagent adopted in the step (3) for the amplification of the treponema TP0136DNA comprises 5 mu L of DNA sample to be detected, 200 mu M dNTPs, 5 mu L of 10 xGoTaq PCR buffer solution, 1.5mM MgCl 2, 0.6 mu M TP0136 primer and 0.5U of hot start Taq PCR polymerase per 50 mu L of PCR amplification reaction reagent;
The condition of the treponema pallidum TP0136DNA amplification in the step (3) is controlled to be 95 ℃ and denatured for 10min; 95 ℃ for 1min, 60 ℃ for 2min and 72 ℃ for 1min, 45 cycles in total; finally, the mixture is extended for 10min at 72 ℃;
the treponema pallidum TP0136DNA sequencing in the step (3) is to carry out the sequencing by a two-dimensional DNA sequencing method through 1.5% agarose gel electrophoresis identification and purification recovery of the PCR amplified product.
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| RU2013102163A (en) * | 2013-01-18 | 2014-07-27 | Федеральное государственное бюджетное учреждение "Государственный научный центр дерматовенерологии и косметологии" Министерства здравоохранения и Российской Федерации (ФГБУ "ГНЦДК" Минздрава России) | COMPLEX OF TREPONEMA PALLIDUM SPECIFIC ANTIGENS FOR USE IN IMMUNOSEROLOGICAL STUDIES IN THE DIAGNOSIS OF SYPHILIS |
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| Title |
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| Wujian Ke等.Treponema pallidum subsp. pallidum TP0136 Protein Is Heterogeneous among Isolates and Binds Cellular and Plasma Fibronectin via its NH2-Terminal End.PLOS Neglected Tropical Diseases.2015,图1，第10页结果第1部分. * |
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