CN111593132A - 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 the treponema pallidum. The method is used for distinguishing different subtypes of the treponema pallidum by detecting the heterogeneity of TP0136 genes of different strains of the treponema pallidum, and makes up the defects of a syphilis molecular typing detection method; has the characteristics of exact 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.
Background
Syphilis is a sexually transmitted disease caused by treponema pallidum infection, and is the third infectious disease of type B in our country. The innate spread of treponema pallidum is the leading cause of fetal and perinatal death in developing countries. Early mucosal damage (chancroid) caused by treponema pallidum infection provides entry of HIV into the human portal, increasing the risk of infection and transmission of HIV, and posing a serious threat to public health. Therefore, the effective prevention and control of treponema pallidum infection is imminent. The treponema pallidum molecular typing system is an important means for developing the epidemiological research of treponema pallidum molecules, is helpful for explaining the relationship between the genotype and the virulence and the invasiveness of the strain, can evaluate the relationship between the genotype and the disease course stage, the disease outcome and the clinical drug resistance, and provides a basis for formulating the control strategy and the effect evaluation of the treponema pallidum.
Dividing the arp into 21 subtypes of 2-22 by the research team of the American disease control center according to the difference of the number of arp gene repeated segments among treponema pallidum strains; tpr is divided into 16 subtypes of a-p according to the RFLP map after the tpr EGJ gene is enzyme-cut, and a treponema pallidum molecular typing method is created and is commonly called CDC typing. Then, Marra et al adopted sequencing technology according to TP0548 gene heterogeneity to divide TP0548 gene into 9 subtypes a-i. Katz et al classified rpsA into 4 subtypes of 8-11 according to the difference in the number of repeats of rpsA gene G. Due to the poor efficiency of rpsA typing, more research teams currently choose to combine CDC and TP0548 typing methods, called new typing methods. In recent years, researchers have analyzed multiple variable sites and tried to select new genetic sites such as TP0279, TP0558, TP0326 and the like for clinical identification, but the typing efficiency is not ideal. Therefore, the existing treponema pallidum typing method cannot meet the clinical diagnosis requirement of syphilis, cannot distinguish the molecular type of the treponema pallidum in nearly 90% syphilis infected patients in China, cannot be accurately used for the research of the molecular epidemiology of the syphilis in China and the clinical evaluation, and has urgent clinical need of a more effective treponema pallidum molecular typing method.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the defects of the prior art, the molecular typing method which is rapid, accurate, simple and convenient and is suitable for distinguishing different strains of the treponema pallidum is provided so as to facilitate the differential diagnosis of the treponema pallidum infection and the research of molecular epidemiology. Our previous studies found that there was genetic heterogeneity of TP0136 in different strains of Treponema pallidum. By utilizing the characteristic, a novel treponema pallidum molecular typing method is established by researching the heterogeneity of TP0136 genes in different strains of the treponema pallidum. The invention utilizes the gene heterogeneity of different strains of the treponema pallidum TP0136, establishes a new TP0136 molecular typing method based on molecular biology technology, is combined with the traditional three-gene typing method, and contributes to the molecular epidemiological research of the treponema pallidum, reveals the correlation between the molecular typing and the clinical characteristics of the treponema pallidum and further improves the molecular typing system of the traditional treponema pallidum.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity, which is established by utilizing the heterogeneity of treponema pallidum TP0136 protein; the method comprises the following specific steps: passage and extraction of treponema syphilis strain; analyzing a Treponema pallidum TP0136 sequence and designing a primer; amplification, sequencing and typing of the treponema TP0136 DNA.
Further, the treponema pallidum comprises 9 treponema pallidum and 23 treponema pallidum clinical isolates separated from clinical syphilis patients in Guangdong province; the 9 strains of Treponema pallidum with syphilis comprise a NicholsHouston strain, a Nichols Seattle strain, a Nichols Dallas strain, a DAl-1 strain, a Bal73-1 strain, a Seattle81-4 strain, a Chicago strain, a MexicoA strain and an SS14 strain.
Further, the passage of the Treponema pallidum strain described in step (1) is performed in the testis of New Zealand white rabbits.
Further, the 1 Xcell lysis buffer used in the extraction of the treponema strain described in step (1) contains 10mM Tris, 0.1M EDTA, 0.5% SDS; the 2 Xcell lysis buffer contained 20mM Tris-HCl,0.2MEDTA, 1% SDS.
Further, the primer sequence used in amplification of the treponema TP0136DNA in the step (3) is shown as SEQ ID NO: 1-3.
Further, Treponema pallidum TP0136 described in step (3)The PCR amplification reaction reagent used in DNA amplification comprises 5 mul of DNA sample to be detected, 200 mul dNTPs, 5 mul 10 × Go Taq PCR buffer solution and 1.5mM MgCl for every 50 mul PCR amplification reaction reagent20.6. mu.M TP0136 primer, 0.5U of hot start Taq PCR polymerase.
Further, the amplification condition of the treponema TP0136DNA in the step (3) is controlled to be 95 ℃ for 10 min; 45 cycles of 95 ℃ for 1min, 60 ℃ for 2min and 72 ℃ for 1 min; finally, extension is carried out for 10min at 72 ℃.
Further, the sequencing of the treponema TP0136DNA in the step (3) is to perform 1.5% agarose gel electrophoresis identification, purification and recovery on a PCR amplified product, and perform sequencing by a bidirectional DNA sequencing method.
Compared with the prior art, the invention has the following beneficial effects:
the method is used for distinguishing different subtypes of the treponema pallidum by detecting the heterogeneity of TP0136 genes of different strains of the treponema pallidum, and makes up the defects of a syphilis molecular typing detection method; has the characteristics of exact detection result and high sensitivity, and is easy to popularize.
Drawings
FIG. 1 is an electrophoretogram of PCR product of the TP0136 gene of Treponema pallidum;
wherein, the left side is amplification product of syphilis TP0136 gene; the right side is the DNA molecular weight standard.
FIG. 2 is a graph showing the typing results of the TP0136 gene of Treponema pallidum;
wherein, the gene is divided into 6 subtypes according to the heterogeneity of the treponema pallidum TP0136 gene, wherein the gene sequences of NicholsHouston, Bal73-1 and Chicago are consistent and are divided into a first type; the gene sequences of Nichols Dallas and Nichols Seattle are consistent and divided into the second type; the other four types are respectively Dal-1, Seattle81-4, SS14 and Mexico A in sequence.
Detailed Description
A molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity, which is established by utilizing the heterogeneity of treponema pallidum TP0136 protein; the method comprises the following steps: passage and extraction of treponema syphilis strain; analyzing a Treponema pallidum TP0136 sequence and designing a primer; amplification, sequencing and typing of the DNA of the treponema TP 0136. The method comprises the following specific steps:
(I) passage and culture of New Zealand white rabbit testis of Treponema pallidum:
passage and isolation of the 9 Treponema pallidum in New Zealand white rabbit testis: 2mL of frozen 9 Treponema pallidum strains with the concentration of 1X 108 Tp/mL (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) are inoculated into New Zealand white rabbit testis, and 1 mL of each testis is inoculated. 9 Treponema pallidum syphilis proliferates in the testis of New Zealand white rabbits by intratesticular inoculation of the New Zealand white rabbits. Before infection experiments were performed, all new zealand white rabbits were tested by Venereal Disease Research Laboratory (VDRL) and fluorescent treponema antibody adsorption (FTA-ABS) to rule out rabbit treponema t. Only VDRL and FTA-ABS negative animals were used for this experiment. The rabbit testis is checked whether orchitis appears every day, and blood is drawn every three days to detect VDRL and FTA-ABS indexes in serum. After the New Zealand white rabbits form orchitis and VDRL and FTA-ABS indexes in serum are changed to positive, the New Zealand white rabbits are euthanized, and then the testis is cut off under aseptic operation, cut up and centrifuged to prepare bacterial suspension of 1 multiplied by 108 Tp/ml.
(II) extracting the DNA of the treponema pallidum strain:
extraction of 9 treponema pallidum strain DNAs: separating spirochete from rabbit tissue fragment by low speed centrifugation, and centrifuging to precipitate treponema pallidum with micro centrifuge. Tp was resuspended in 1 Xcell lysis buffer (10 mM Tris, 0.1M EDTA, 0.5% SDS). Resuspend Tp and freeze at-20 ℃ or extract DNA using QIAamp DNA Mini kit (QIAGEN) as required. The method comprises the following specific steps: 1) pipetting 20. mu.L QIAGEN protease (or proteinase K) to the bottom of a 1.5mL centrifuge tube; 2) adding 200 mu L of a sample of the genome to be extracted into a centrifuge tube; 3.) add 200. mu.L of buffer AL to the sample and mix by vortexing for 15 seconds. Complete mixing to obtain a homogenous solution is important to ensure adequate lysis of the sample. If the sample volume is greater than 200. mu.L, please increase the amount of protease and buffer AL equally. In practice, please note that QIAGEN protease (or proteinase K) is not added directly to the buffer AL; 4) the DNA yield reaches the maximum value under the condition that the incubation is carried out for 10 minutes at 56 ℃, and the yield cannot be further improved by prolonging the incubation time; 5) performing rapid centrifugation to remove liquid drops remained in the cover of the 1.5mL centrifuge tube; 6) add 200. mu.L (sample equal volume) of ethanol (96-100%) and mix by vortexing for 15 seconds. After the oscillation is finished, quickly centrifuging to remove liquid drops remained in the cover of the 1.5mL centrifuge tube; 7; the mixture obtained in step 6 was transferred to a QIAamp Mini centrifuge column (in a 2mL collection tube). The caps were closed (closing the cap of each centrifuge tube during centrifugation to prevent aerosol generation during centrifugation) and centrifuged at 6000g for 1 minute. Placing the QIAamp Min spin column into a new clean 2mL receiving tube, and discarding the filtrate together with the used collection tube; 8) the QIAamp Mini spin column was carefully opened and 500. mu.L of buffer AW1 was added (care was taken not to wet the edge ring). The lid was closed and centrifuged at 6000g for 1 min. The QIAamp Mini spin column 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 the step; 9) the filtrate was discarded, the QIAamp Mini spin column carefully opened, and 500. mu.L of buffer AW2 was added (care was taken not to wet the edge ring). The cover is tightly covered, and the centrifuge is carried out for 3 minutes at the maximum rotation speed of 20000 g; 10) the QIAamp Mini spin column was transferred to a new 2mL collection tube. The centrifuge is carried out at a maximum speed of 20000g for 1 minute. This step can help reduce the likelihood of buffer AW2 remaining; 11) the QIAamp Mini spin column was transferred to a new 1.5mL collection tube. Carefully open the column and add 200 μ L of buffer AE or double distilled water (elution volumes greater than 200 μ L are not suitable for a 1.5mL collection tube because the lower edge of the column is in contact with the eluate, which may result in aerosol formation during centrifugation; elution volumes less than 200 μ L significantly increase the DNA concentration in the eluate, but reduce the overall DNA yield; for less than 1 μ g of DNA, 50 μ L of eluate is recommended; 2 washes with 100 μ L of eluate are nearly as effective as one wash with 100 μ L of eluate). Incubate at room temperature (15-25 ℃) for 1 minute (generally, an extension of the incubation time to 5 minutes increases the yield) and then centrifuge at 6000g for 1 minute. If 200. mu.L of buffer AE is added to perform secondary elution, the yield can be improved by about 15%. After extraction the DNA was stored at-20 ℃. DNA was extracted for PCR detection and post amplification manipulation. After extraction the DNA was stored at-20 ℃.
(III) sequence analysis and primer design of treponema TP 0136:
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 protein and DNA databases, and 9 Treponema pallidum (Nichols Houston, Nichols Seattle, Nichols Dallas, DAl-1, Bal73-1, Seattle81-4, Chicago, Mexicoa and SS14) of the genus Treponema pallidum were subjected to multiple sequence alignment using BioEdit7.1 software (http:// biodata.
Primer design for treponema TP 0136: primer design software was applied: the Primer3: WWW Primer (http:// biolols. umapplied. edu/bioapps/Primer 3-www.cgi) was used to perform Primer design on the gene sequence of Nichols standard strain TP0136 in GenBank query. The TP0136 signal peptide was predicted using SignalP4.1Server software (http:// www.cbs.dtu.dk/services/SignalP /), and the TP0136 open reading frame primer contained no signal peptide.
(IV) amplification, sequencing and typing of Treponema pallidum TP0136 DNA:
amplification of treponema pallidum TP0136 DNA: TP0136DNA of 9 Treponema pallidum strains of Treponema pallidum is amplified by conventional PCR. 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 size of the amplification product is 196 bp. The type 2 and type 3 upstream primers are 5'-CGCACGCCGCACGTCTATTT-3' (SEQ ID No: 3); the type 2 and 3 downstream primers were: 5'-CAGCGGAGGGACCAGCAGCA-3' (SEQ ID No:2), the amplification 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 sizes of the amplified products are 196bp respectively. The type 5 and type 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 amplification products are all 196bp in size. 50 μ L of PCR amplification reaction reagent 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 deg.C for 10 min; 45 cycles of 95 ℃ for 1min, 60 ℃ for 2min and 72 ℃ for 1 min; finally, extension is carried out for 10min at 72 ℃.
Sequencing and typing of treponema pallidum TP0136 amplification products: the amplified PCR products were identified by electrophoresis on a 1.5% agarose gel (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 dNTPs and primers present in the PCR product mixture that are not consumed will affect the subsequent reaction. ExoSAP-IT utilizes two hydrolases, Exonuclease Exonase I and Shrimp alkaline phosphatase stretch Alkalinephosphospatase, along with specially formulated buffers, to remove unwanted dNTPs and primers from the PCR product. Exonuclease I removes the remaining single stranded primers and any extraneous single stranded DNA generated in the PCR. Shrimp alkaline phosphatase removes residual dNTPs from the PCR mixture, which may affect subsequent reactions. ExoSAP-IT was added directly to the PCR product and incubated at 37 ℃ for 15 minutes. The enzyme is active in the buffer system of the PCR and therefore does 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. Using ExoSAP-IT, both short and long PCR products gave 100% yield. Sequencing ORF of Treponema pallidum TP0136 by using a bidirectional DNA sequencing method, and performing multiple sequence comparison on sequencing results by using BioEdit7.1 software to obtain typing results (shown in figure 2 specifically).
(V) validation of Treponema pallidum TP0136 typing capacity:
the treponema pallidum TP0136 typing method is applied to typing 23 treponema pallidum clinical isolates from southern medical university skin disease hospital. The results show that the types of the 23 treponema pallidum clinical isolates are all 100% located in treponema pallidum subspecies of treponema, which indicates that the method has high specificity. Wherein 4 treponema pallidum clinical isolates (GD003, GD008, GD022 and GD023) are type I; the 16 treponema pallidum clinical isolates (GD001, GD005, GD006, GD007, GD010, GD011, GD012, GD013, GD014, GD015, GD016, GD017, GD018, GD019, GD020 and GD021) are type 5; the 3 treponema pallidum clinical isolates (GD002, GD004 and GD009) are of type 6.
Even if the Arp/Tpr/TP0548 triple genotyping method is used, only 23 clinical isolates of Treponema pallidum can be classified into type 5 (13D/D, 14D/f, 14D/g, 15D/f, 16A/e); however, if the novel treponema TP0136 molecular typing method is combined with the traditional three-gene typing, 23 clinical isolates in the research can be further divided into 9 types (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.
Therefore, the kit has high specificity (100%), can be used for distinguishing spirochete infection of different treponema, and is beneficial to differential diagnosis of clinical pathogens; but also can improve the sensitivity of traditional genotyping, improve the genotyping efficiency by 180 percent and is beneficial to the research of clinical epidemiology level.
Claims (9)
1. A molecular typing method for distinguishing different subtypes of treponema pallidum based on TP0136 gene heterogeneity, which is characterized in that the method is established by utilizing the TP0136 protein heterogeneity of the treponema pallidum.
2. The molecular typing method for distinguishing the different subtypes of treponema pallidum based on the TP0136 gene heterogeneity according to claim 1, which comprises the following steps:
(1) passage and extraction of treponema syphilis strain;
(2) analyzing a Treponema pallidum TP0136 sequence and designing a primer;
(3) amplification, sequencing and typing of the DNA of the treponema TP 0136.
3. The molecular typing method for differentiating the different subtypes of treponema pallidum based on the TP0136 gene heterogeneity according to claim 2, wherein the genus treponema pallidum includes 9 treponema pallidum and 23 clinical isolates of treponema pallidum isolated from clinical patients with treponema pallidum in Guangdong province; the 9 strains of Treponema pallidum with syphilis comprise a Nichols Houston strain, a Nichols Seattle strain, a Nichols Dallas strain, a DAl-1 strain, a Bal73-1 strain, a Seattle81-4 strain, a Chicago strain, a MexicoA strain and an SS14 strain.
4. The molecular typing method for differentiating different subtypes of treponema pallidum based on the heterogeneity of TP0136 gene as claimed in claim 2, wherein the passaging of the treponema pallidum strain in the step (1) is performed in testis of New Zealand white rabbit.
5. The molecular typing method for differentiating the different subtypes of treponema pallidum based on the TP0136 gene heterogeneity according to claim 2, wherein the 1X cell lysis buffer used in the extraction of the treponema strain in the step (1) comprises 10mM Tris, 0.1M EDTA, 0.5% SDS; the 2 Xcell lysis buffer contained 20mM Tris-HCl,0.2M EDTA, 1% SDS.
6. The molecular typing method for distinguishing the different subtypes of treponema pallidum based on the heterogeneity of TP0136 gene as claimed in claim 2, wherein the primer sequences used in the amplification of the treponema TP0136DNA in step (3) are shown in SEQ ID NO 1-3.
7. The molecular typing method according to claim 2, wherein the PCR amplification reagents used in the amplification of the TP0136DNA of Treponema pallidum in step (3) comprise 5. mu.L of the DNA sample to be detected, 200. mu.M dNTPs, 5. mu.L 10 × GoTaq PCR buffer, and 1.5mM Mg per 50. mu.L of the PCR amplification reagentsCl20.6. mu.M TP0136 primer, 0.5U of hot start Taq PCR polymerase.
8. The molecular typing method for differentiating different subtypes of treponema pallidum based on the heterogeneity of TP0136 gene as claimed in claim 2, wherein the amplification condition of the treponema pallidum TP0136DNA in the step (3) is controlled to denature at 95 ℃ for 10 min; 45 cycles of 95 ℃ for 1min, 60 ℃ for 2min and 72 ℃ for 1 min; finally, extension is carried out for 10min at 72 ℃.
9. The molecular typing method for distinguishing the different subtypes of treponema pallidum based on the TP0136 gene heterogeneity according to claim 2, wherein the sequencing of the TP0136DNA of the treponema pallidum in the step (3) is performed by identifying, purifying and recovering the PCR amplified product through 1.5% agarose gel electrophoresis, and sequencing through the two-dimensional DNA sequencing method.
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| 马虎;余先华;张仲毅;: "梅毒螺旋体基因分型的相关性研究" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116042678A (en) * | 2023-02-08 | 2023-05-02 | 南方医科大学皮肤病医院（广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心） | Fusion protein for detecting nerve syphilis and kit thereof |
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