A rapid and sensitive PCR strategy employed for amplification and sequencing of porA from a single colony-forming unit of Neisseria meningitidis

Saunders, Nancy B.; Zollinger, Wendell D.; Rao, Venigalla B.

doi:10.1016/0378-1119(93)90001-j

Cited by 52 publications

(47 citation statements)

References 20 publications

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“…The porA gene was sequenced and the corresponding sequence perfectly matched the one previously described. 45 The open reading frame was 1155 bp, close to the size of the wild-type sequence. These features promoted the PorA expression as inclusion bodies.…”

Section: Resultsmentioning

confidence: 81%

“…The porA gene was amplified by PCR using DNA from N. meningitidis B:15:P1.3 as the template 45 and Pfu DNA polymerase (Stratagene). The strain has been annotated as B15: P1.7b,3, following the PorA VR type designation, 46 and the original PorA gene sequence has been deposited 45 at the GenBank as acc # L02929.…”

Section: Resultsmentioning

confidence: 99%

“…The strain has been annotated as B15: P1.7b,3, following the PorA VR type designation, 46 and the original PorA gene sequence has been deposited 45 at the GenBank as acc # L02929. This strain belongs to the clonal complex group ET-5.…”

Section: Resultsmentioning

confidence: 99%

“…Both primers (sequences displayed in Table 1) were designed using the PorA gene sequence described by Saunders et al 45 PCR included the following steps: 94 C for 15 sec, 55 C for 30 sec and 68 C for 90 sec, over 30 cycles. The amplified porA gene included NdeI and HindIII restrictions sites at its 5 0 and 3 0 ends, respectively, as they were introduced into the primer sequences.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Oral administration of recombinantNeisseria meningitidisPorA genetically fused toH. pyloriHpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

Vásquez

Manzo

Soto

et al. 2015

Human Vaccines & Immunotherapeutics

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Section: Resultsmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Oral administration of recombinantNeisseria meningitidisPorA genetically fused toH. pyloriHpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

Vásquez

Manzo

Soto

et al. 2015

Human Vaccines & Immunotherapeutics

View full text Add to dashboard Cite

“…To date, the characterization of meningococci has relied on serogrouping, serotyping and serosubtyping using serological methods, thereby limiting the designation of serotypes and serosubtypes because a panel of mAbs is used. Newer nucleotide sequence methods, based on the detection and sequencing of the porB and porA genes for serotype (class 2/3 OMP) and serosubtype (class 1 OMP), respectively, have led to a better understanding of the variability of these proteins (Urwin et al, 1998a, b, c;Clarke et al, 2001a;Jelfs et al, 2000;Molling et al, 2000;Feavers et al, 1996;Saunders et al, 1993;Smith et al, 1995). However, most of these methods are only useful when N. meningitidis has been isolated from blood or cerebrospinal fluid (CSF) and they therefore rely on the availability of a culture (Jelfs et al, 2000;Feavers et al, 1996).…”

mentioning

confidence: 99%

Detection and genotyping of meningococci using a nested PCR approach

Diggle

Clarke

2003

Journal of Medical Microbiology

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An effective vaccine against Neisseria meningitidis serogroup B is required. Outer-membrane protein vaccines have been developed, which may provide protection against common circulating serotypes and serosubtypes in some countries. However, limited genosubtyping data are available because most laboratories use mAbs directed against a limited number of specific serotypes and serosubtypes and laboratories do not genosubtype directly from body fluids due to the lack of a sensitive PCR method. A nested PCR was therefore developed that enables the amplification of the porA gene directly from clinical samples and has the required sensitivity for nucleotide sequencing of the three main variable regions, VR1, VR2 and VR3. Data were compared with those from culturebased nucleotide sequencing, and the use of this method increased the availability of genosubtype information by 45 %, thereby indicating the impact that this methodology has on the data provided and the implications for vaccine design. INTRODUCTIONMeningococcal disease remains a significant public health problem, and a vaccine against Neisseria meningitidis serogroup B is urgently needed (Rosenstein et al., 2001). However, the use of a capsule-based vaccine is unlikely because the meningococcal polysaccharide is identical to a human carbohydrate [AE (2 ! 8) N-acetyl neuraminic acid or polysialic acid] (Finne et al., 1987;Hayrinen et al., 1995). Vaccines based on other antigens therefore require development and the outer-membrane proteins (OMPs) of the meningococcus have been investigated (Rosenstein et al., 2001;Al'Aldeen & Cartwright, 1996). Although OMP-based vaccines are not the solution for combating serogroup B meningococcal infection, they may be useful in some circumstances (Al'Aldeen & Cartwright, 1996;Rappuoli, 2001). However, such proteins are highly antigenically variable, and an OMP vaccine cannot protect against all serotypes and serosubtypes of N. meningitidis. Therefore, selected OMPs must be used, based on the prevalent serotypes and serosubtypes in a given population. However, to develop such vaccines, high-quality data must be gained to determine such prevalence. To date, the characterization of meningococci has relied on serogrouping, serotyping and serosubtyping using serological methods, thereby limiting the designation of serotypes and serosubtypes because a panel of mAbs is used. Newer nucleotide sequence methods, based on the detection and sequencing of the porB and porA genes for serotype (class 2/3 OMP) and serosubtype (class 1 OMP), respectively, have led to a better understanding of the variability of these proteins (Urwin et al., 1998a, b, c;Clarke et al., 2001a;Jelfs et al., 2000;Molling et al., 2000;Feavers et al., 1996;Saunders et al., 1993;Smith et al., 1995). However, most of these methods are only useful when N. meningitidis has been isolated from blood or cerebrospinal fluid (CSF) and they therefore rely on the availability of a culture (Jelfs et al., 2000;Feavers et al., 1996). Other methods rely on sufficient numbers of me...

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Immunoproteomic identification of the hypothetical protein NMB1468 as a novel lipoprotein ubiquitous in Neisseria meningitidis with vaccine potential

Hsu

Lin

et al. 2008

Proteomics

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Many potential vaccine candidates for serogroup B Neisseria meningitidis (NMB) have been identified by reverse vaccinology, a genome-based approach. However, some candidates may be unseen owing to uncertain annotation or their peculiar properties. In this study, we describe the preparation and identification of a novel lipoprotein expressed in all meningococcal strains tested. mAb were first prepared from mice immunized with a meningococcal B strain isolated in Taiwan. Total proteins from the immunizing strain were separated by 2-DE in duplicate. Clone 4-7-3, which crossreacted to 174 tested meningococcal isolates, was used as the primary antibody for Western blotting. The immunoreactive spot was identified by LC-mass spectrometric analysis of the corresponding spot from the silver-stained gel and confirmed by molecular biology approach to be a novel lipoprotein encoded by the hypothetical NMB1468 gene. The potential use of this protein, designated Ag473/NMB1468, as a vaccine component was evaluated using the recombinant protein produced in Escherichia coli. Immunized mice were found to be protected from developing meningococcal disease after intraperitoneal inoculation with a lethal dose of meningococcal strain Nm22209, suggesting that Ag473/NMB1468 may be a promising vaccine candidate. This study also demonstrates the usefulness of the immunoproteomic approach in identification of novel vaccine candidates.

show abstract

A rapid and sensitive PCR strategy employed for amplification and sequencing of porA from a single colony-forming unit of Neisseria meningitidis

Cited by 52 publications

References 20 publications

Oral administration of recombinantNeisseria meningitidisPorA genetically fused toH. pyloriHpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

Oral administration of recombinantNeisseria meningitidisPorA genetically fused toH. pyloriHpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

Detection and genotyping of meningococci using a nested PCR approach

Immunoproteomic identification of the hypothetical protein NMB1468 as a novel lipoprotein ubiquitous in Neisseria meningitidis with vaccine potential

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