Objective To assess the sensitivity,specificity,and feasibility of 4 recombinant Treponema pallidum antigen-based rapid tests in the diagnosis of syphilis.Methods A total of 970 outpatients were selected from the Sexu...Objective To assess the sensitivity,specificity,and feasibility of 4 recombinant Treponema pallidum antigen-based rapid tests in the diagnosis of syphilis.Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital.Venous blood was collected and serum was extracted.T.pallidum antibodies in whole blood,anticoagulant whole blood,and serum were detected using 4 recombinant T.pallidum antigen-based rapid tests.T.pallidum haemagglutination test(TPHA) was considered as the gold standard for the detection of T.pallidum specific antibodies in serum.The sensitivities and specificities of four methods were analyzed.Results The sensitivities and specificities of Abbott Determine Syphilis TP test,SD-BIOLINE Syphilis 3.0 test,VISITECT-SYPHILIS test,and Syphicheck-WB test for serum specimens were 100% and 98.9%,95.7% and 98.0%,94.6% and 98.2%,68.1% and 98.9%;for whole blood were 74.1% and 99.5%,87.9% and 99.4%,73.2% and 99.7%,64.7% and 99.7%.The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA(P>0.05).Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis.Furthermore,they are more sensitive for serum specimens than whole blood.展开更多
To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. ...To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.展开更多
To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was ...To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.展开更多
The Gpd gene was amplified from the genomic DNA of Treponema pallidum and cloned into the appropriate site of pcDNA3.1(+) vector. The expression of pcDNA3.1(+)-Gpd in HeLa cells was tested with Western blotting and te...The Gpd gene was amplified from the genomic DNA of Treponema pallidum and cloned into the appropriate site of pcDNA3.1(+) vector. The expression of pcDNA3.1(+)-Gpd in HeLa cells was tested with Western blotting and technology of immunocytochemistry. New Zealand rabbits were immunized with the eukaryotic expression recombinant pcDNA3.1(+)-Gpd. A fusion protein of Gpd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected by Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1∶1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls (P<0.01). All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.展开更多
Objective: To obtain recombinant Treponema pallidum subsp, pallidum (TP 17KD) lipoprotein in large quantities by amplification and to further purify antigens for laboratory diagnosis of syphilis and development of a s...Objective: To obtain recombinant Treponema pallidum subsp, pallidum (TP 17KD) lipoprotein in large quantities by amplification and to further purify antigens for laboratory diagnosis of syphilis and development of a syphilis vaccine. Method: The Tppl7 lipoprotein gene was amplified from the TP(strain Nichols), and then it was recombinated into a plasmid pMAL-2c and cloned within E. coli 12-TB1. The host bacteria containing recombinant plasmids were induced with IPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double digestion and PCR. Recombinant plasmids were transformed into E. coli and the E. coli carrying recombinant plasmids were induced. The expression of TP 17KD was detected by sodium dedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showed that the induced E. coli carrying recombinant plasmid could produce 60KD fusion protein at high levels. Gel scanning showed that 17KD protein expression in E. coli accounted for 10 % of total cellular protein. The recombinant protein antigen reacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developing new techniques of laboratory diagnosis for syphilis and new vaccines. Preliminary clinical application showed that the fusion protein could be used for the diagnosis of syphilis.展开更多
Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Met...Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.展开更多
Objective: To evaluate the clinical application of multiplex PCR in the detection of Treponema pallidum, Herpes simplex virus (HSV), and Haemophilus ducreyi. Method: Three standard strains were used to set up a multip...Objective: To evaluate the clinical application of multiplex PCR in the detection of Treponema pallidum, Herpes simplex virus (HSV), and Haemophilus ducreyi. Method: Three standard strains were used to set up a multiplex PCR (MPCR) for detecting syphilis, herpes genitalis, and chancroid simultaneously. Samples from 122 patients with genital ulcer disease(GUD) were subjected to MPCR and the results were compared with these of dark-fidd microscopy and TP serology, HSV anligen ELISA,and H. ducreyi culture, Result: In the 122 patients with GUD, MPCR identified 34 casesof T.pallidum infection, 40 cases of HSV infection, and 2 cases of mixed infection of T.pallidum and herpes. No positive results of H. ducreyi were found. The sensitivity of MPCR to T. pallidum and herpes was 100% and 93.3%, respectivdy. The sensitivities of dark-field microscopy and TP serology, HSV antigen ELISA, and H. ducreyi culture was 35.3%, 50% and 100%, respectively. Conclusion: MPCR showed a relatively higher sensitivity for T.pallidum as compared with the routine techniques. Although its sensitivity for HSV was not as good as that of antigen ELISA, it also yielde da high detection rate. MPCR can detect more than one pathogen. It is simple, quick, sensitive, and suitable for clinical use or epidemiological investigation.展开更多
Screening for maternal syphilis has been an essential component of routine antenatal screening tests in most countries for many years. This is not only because of the virulence of the spirochete which causes the infec...Screening for maternal syphilis has been an essential component of routine antenatal screening tests in most countries for many years. This is not only because of the virulence of the spirochete which causes the infection but also because of its vertical transmission rate and the potential severe adverse complications/morbidity that can result from its transmission to the fetus. Although the incidence of maternal syphilis and its fetal sequalae in low-income countries has been considerable for several years, the disease has been almost non-existent in high income countries with wide antenatal screening coverage and effective treatment programmes for Syphilis. The recent alarming increase in the incidence of maternal syphilis in high income countries has spawned a renewed public health interest in the infection, with several countries updating and strengthening public health guidance in an attempt to stem this dramatic trend. This is a short clinical update for the practising obstetrician on how to manage the antenatal patient with a positive syphilis screening test.展开更多
基金Supported by the WHO project on rapid diagnosis of syphilis (RFA-SDI-2001-02)
文摘Objective To assess the sensitivity,specificity,and feasibility of 4 recombinant Treponema pallidum antigen-based rapid tests in the diagnosis of syphilis.Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital.Venous blood was collected and serum was extracted.T.pallidum antibodies in whole blood,anticoagulant whole blood,and serum were detected using 4 recombinant T.pallidum antigen-based rapid tests.T.pallidum haemagglutination test(TPHA) was considered as the gold standard for the detection of T.pallidum specific antibodies in serum.The sensitivities and specificities of four methods were analyzed.Results The sensitivities and specificities of Abbott Determine Syphilis TP test,SD-BIOLINE Syphilis 3.0 test,VISITECT-SYPHILIS test,and Syphicheck-WB test for serum specimens were 100% and 98.9%,95.7% and 98.0%,94.6% and 98.2%,68.1% and 98.9%;for whole blood were 74.1% and 99.5%,87.9% and 99.4%,73.2% and 99.7%,64.7% and 99.7%.The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA(P>0.05).Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis.Furthermore,they are more sensitive for serum specimens than whole blood.
基金This work was supported by Hunan Provincial Education Department (002A046) and Department of Public Health of Hunan province (B2003 085)
文摘To construct the recombinant plasmid of eukaryotic expression containing Tp92 gene from Treponema pallidum and study its immunogenicity in New Zealand white rabbits. Tp92 gene was amplified from the genomic DNA of T. pallidum by polymerase chain reaction (PCR) and subcloned into appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzyme digestion, the recombinant plasmid was transfected into HeLa cells using liposome, and the expressed protein was identified by immunocytochemistry and Western blotting. After verifying that the Tp92 antigen gene fragment could be expressed in HeLa cells, 100?μg of recombinant plasmids [pcDNA3.1(+)-Tp92], 100 μg of control plasmids [pcDNA3.1(+)] or 0.5 ml PBS buffer were administered in 3 groups of New Zealand white rabbits (6 rabbits/group), and the booster immunizations were employed at 2-week interval for 3 times. ELISA assay was used for the quantitative detection of the specific antibody in the sera of rabbits, and the proliferation response of spleen cells was detected by MTT assay. It was found that the target gene Tp92 segment about 2103 bp was obtained, and the DNA sequence of Tp92 gene constructed in pcDNA3.1 (+) vector was consistent with the published nucleotide sequence. The homologies of the nucleotide and putative amino acid sequences of Tp92 gene between T.pallidum subsp. pallidum Nichols and various pathogenic treponeme strains were 95.5%-100%. The analysis of immunocytochemistry and Western blotting showed that Tp92 gene segment constructed in pcDNA3.1(+) vector could express a fusion protein with a calculated molecular mass of 77 kDa in HeLa cells and the expressed protein could react with positive blood serum from syphilis patient. The specific antibody IgG titers were observed and the highest titer was 1∶1024 in rabbits after 3 times with pcDNA3.1(+)-Tp92. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1(+) ( P <0.05). The successful expression of the eukaryotic expression plasmid of Tp92 gene from T. pallidum was obtained in eukaryotic system and strong responses of humoral and cellular immunity was evoked by DNA vaccine of pcDNA3.1(+)-Tp92 in rabbits thus establishing a solid basis for the future studies in the biological activities and for the development of the syphilis DNA vaccine.
文摘To clone and express the recombinant outer membrane protein Tp0453 of Treponema pallidum and to analyze the immuno-reactivity and immunogenicity of the expressed protein, the immuno-dominant epitope of the Tp0453 was amplified from the complete genome of T.pallidum by PCR, subcloned into expression vector pQE32 to generate the recombinant plasmid pQE32/Tp0453, then expressed in E.coli M15 and analyzed by SDS/PAGE and Western blotting. The fusion protein expressed was purified with Ni-NTA affinity chromatography. Its immuno-reactivity was assayed by indirect ELISA, and the immunogenicity was determined by immunization with this fusion protein in New Zealand rabbits. In the present study, a fusion protein of molecular weight about 32 kDa was obtained. As demonstrated by Western blotting, the recombinant protein could react specifically with positive IgG sera of patients with syphilis, and the antibodies against T.pallidum in human sera were successfully detected by indirect ELISA. Both the sensitivity and specificity of ELISA based on the Tp0453 fusion protein as were 100% (30/30) when detected with control sera. In comparison with the results of IgG ELISA with those of TPPA. It was found that the sensitivity of ELISA was 96.8% and the specificity was 100%. The difference of ELISA and TPPA was not significant, and the concordance of results between ELISA and TPPA was 98.2%. In addition, specific humoral responses could be elicited by immunization with the recombinant fusion protein in New Zealand rabbits with a specific antibody titer of 1∶1280 after 3 successive doses of immunization. These results demonstrate that the expressed recombinant fusion protein shows excellent immuno-competence and provide foundation to develop a quick diagnostic kid applied to detect the presence of T.pallidum infections.
文摘The Gpd gene was amplified from the genomic DNA of Treponema pallidum and cloned into the appropriate site of pcDNA3.1(+) vector. The expression of pcDNA3.1(+)-Gpd in HeLa cells was tested with Western blotting and technology of immunocytochemistry. New Zealand rabbits were immunized with the eukaryotic expression recombinant pcDNA3.1(+)-Gpd. A fusion protein of Gpd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected by Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1∶1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls (P<0.01). All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.
文摘Objective: To obtain recombinant Treponema pallidum subsp, pallidum (TP 17KD) lipoprotein in large quantities by amplification and to further purify antigens for laboratory diagnosis of syphilis and development of a syphilis vaccine. Method: The Tppl7 lipoprotein gene was amplified from the TP(strain Nichols), and then it was recombinated into a plasmid pMAL-2c and cloned within E. coli 12-TB1. The host bacteria containing recombinant plasmids were induced with IPTG. The Tpp 17KD lipoprotein gene was amplified by us-ing PCR and positive clones were screened with double digestion and PCR. Recombinant plasmids were transformed into E. coli and the E. coli carrying recombinant plasmids were induced. The expression of TP 17KD was detected by sodium dedecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot. Results:Gel staining with Coomassie blue G-250 showed that the induced E. coli carrying recombinant plasmid could produce 60KD fusion protein at high levels. Gel scanning showed that 17KD protein expression in E. coli accounted for 10 % of total cellular protein. The recombinant protein antigen reacted with the sera of syphilis patients. Conclusion: Our study lays a cornerstone for developing new techniques of laboratory diagnosis for syphilis and new vaccines. Preliminary clinical application showed that the fusion protein could be used for the diagnosis of syphilis.
基金Focal point financial assistance entry(002A046)Department of Education, Hunan Province Scientific Research Foundation(B2003-085) Department of Public Health, Hunan province.
文摘Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.
文摘Objective: To evaluate the clinical application of multiplex PCR in the detection of Treponema pallidum, Herpes simplex virus (HSV), and Haemophilus ducreyi. Method: Three standard strains were used to set up a multiplex PCR (MPCR) for detecting syphilis, herpes genitalis, and chancroid simultaneously. Samples from 122 patients with genital ulcer disease(GUD) were subjected to MPCR and the results were compared with these of dark-fidd microscopy and TP serology, HSV anligen ELISA,and H. ducreyi culture, Result: In the 122 patients with GUD, MPCR identified 34 casesof T.pallidum infection, 40 cases of HSV infection, and 2 cases of mixed infection of T.pallidum and herpes. No positive results of H. ducreyi were found. The sensitivity of MPCR to T. pallidum and herpes was 100% and 93.3%, respectivdy. The sensitivities of dark-field microscopy and TP serology, HSV antigen ELISA, and H. ducreyi culture was 35.3%, 50% and 100%, respectively. Conclusion: MPCR showed a relatively higher sensitivity for T.pallidum as compared with the routine techniques. Although its sensitivity for HSV was not as good as that of antigen ELISA, it also yielde da high detection rate. MPCR can detect more than one pathogen. It is simple, quick, sensitive, and suitable for clinical use or epidemiological investigation.
文摘Screening for maternal syphilis has been an essential component of routine antenatal screening tests in most countries for many years. This is not only because of the virulence of the spirochete which causes the infection but also because of its vertical transmission rate and the potential severe adverse complications/morbidity that can result from its transmission to the fetus. Although the incidence of maternal syphilis and its fetal sequalae in low-income countries has been considerable for several years, the disease has been almost non-existent in high income countries with wide antenatal screening coverage and effective treatment programmes for Syphilis. The recent alarming increase in the incidence of maternal syphilis in high income countries has spawned a renewed public health interest in the infection, with several countries updating and strengthening public health guidance in an attempt to stem this dramatic trend. This is a short clinical update for the practising obstetrician on how to manage the antenatal patient with a positive syphilis screening test.
