Expression, immunochemical characterization and localization of the Mycobacterium tuberculosis protein p27.

Rv2108 is a gene of the PPE family of Mycobacterium tuberculosis specific for this bacterial complex and that may encode a putative protein p27. This gene was amplified, inserted into bacterial vectors, sequenced, and expressed as a recombinant protein. Specific antibodies to this protein were generated and used for immunochemical characterization and cellular localization. Mass spectrometric analysis of the expressed protein revealed a molecule that corresponded to the p27 putative protein. The expressed protein was immunologically active, and reacted with antibodies from tuberculosis patient sera. Specific immunoblot analysis confirmed the presence of the p27 antigen in Mycobacterium bovis BCG strain and in human clinical isolates of M. tuberculosis, but not in other mycobacteria tested. Western blot and immunoelectron microscopic analysis of BCG strain indicated that the p27 protein is localized in the membrane of the cell. The specific expression of the p27 protein in the M. tuberculosis complex could provide a novel specific complimentary diagnostic test for the presence of and infection with M. tuberculosis.

Rapid detection of Campylobacter fetus by polymerase chain reaction combined with non-radioactive hybridization using an oligonucleotide covalently bound to microwells.

Campylobacter fetus is recognized as a human and animal pathogen. The isolation and differentiation of C. fetus in diagnostic laboratories is hindered by its relatively slow growth and lack of distinguishing biochemical characteristics. We cloned and sequenced a 1581-bp DNA fragment, IG02, isolated from a C. fetus genomic library. This fragment was used as a probe on DNAs extracted from C. fetus strains and other Campylobacter species: IG02 hybridized only with DNAs from C. fetus strains. A PCR-based test was developed for the detection of C. fetus. A pair of oligonucleotide primers was designed to amplify a 141-bp fragment of IG02. The amplified product was analysed by a non-radioactive sandwich hybridization in microtiter plate using a capture oligonucleotide and a biotin-labelled oligonucleotide for the detection. The combination of PCR and non-radioactive microplate hybridization is a convenient method for the rapid detection of C. fetus.

Cloning of a gene from Mycobacterium tuberculosis coding for a hypothetical 27 kDa protein and its use for the specific PCR identification of these mycobacteria.

PCR targeting the IS 6110 has been considered specific for identification of Mycobacterium tuberculosis and is frequently applied to confirm the presence of this organism directly in biological specimens. However, several authors found that some M. tuberculosis strains failed to hybridize with the IS 6110 probe and other authors found that false-positive results may be obtained for clinical samples when some methods based on IS 6110 are used. In the present study, the p27 gene isolated from a cosmid library was found to be highly specific for M. tuberculosis complex strains and allowed us to develop a PCR-based assay for rapid detection and identification of this mycobacterium. One pair of primers and two oligonucleotide probes were successfully used to amplify and to detect the DNA of strains belonging to the M. tuberculosis complex. These primers and probes did not hybridize with DNA from any of the 21 other mycobacterial species tested. It is worth noting that the chosen primers and probes hybridize with DNA from the M. tuberculosis strain with no IS 6110, furthermore no strain without p27 was found among the 410 strains tested in the present study.

Molecular fingerprinting of Mycobacterium tuberculosis strains isolated in Vietnam using IS6110 as probe.

SETTING: Northern and Southern areas of Vietnam. OBJECTIVE: To study the correlation between DNA fingerprinting of 168 Mycobacterium tuberculosis strains isolated from patients with a particular historical past (political separation of Vietnam for 20 years) and data about geographical origin, drug susceptibility, HIV infection and BCG vaccination status. METHODS: Comparison of restriction fragment length polymorphism (RFLP) patterns produced by Southern hybridization of Pvull-digested chromosomal DNA. RESULTS: The number of IS6110 copies for the 168 strains ranges from 0 to 23. Strains originating from the North or the South differ strongly with respect to the number of copies of IS6110. Indeed, the strains originating from the north have predominantly from 3 to 14 IS6110 copies while the southern strains have predominantly from 15 to 23 IS6110 copies. Furthermore, strains isolated in the North are dispersed into 6 groups whereas 80% of the strains isolated in the South form a single group. Moreover, the prevalence of drug resistance is higher in strains isolated in the South than in the North. No noticeable correlation is observed between RFLP patterns, drug susceptibility, or HIV infection. CONCLUSION: The IS6110 fingerprints of 168 M. tuberculosis strains isolated in Vietnam showed a high range of polymorphism. Only a few strains have been found with no IS6110 (1.8%). The differences between the strains from the North and South, having more than six IS6110, suggests that they derived from ancestral strains that would be distinguishable by the number of IS6110 and their transposition sites throughout the genome. The genomic structure of the population of strains from South Vietnam resembles that of the Beijing strain population. This could account for a similar evolution of M. tuberculosis due to a selection by BCG-induced immunity in the two populations.

A simple and reliable method for the detection of the 30delG mutation of the CX26 gene.

Mutations in the CX26 gene (GJB2), encoding the gap-junction protein Connexin-26, have been shown to be the major cause of non-syndromic recessive deafness. Among these mutations, the deletion of a guanine within the stretch of six G between nucleotide positions +30 and +35 of the CX26 cDNA (30delG) accounts for the majority of this kind of deafness. Molecular detection of the 30delG mutation is usually performed by direct sequencing analysis of PCR products or by SSCP. To detect this mutation we developed an easy and reliable method, based on PCR, followed by a non-radioactive sandwich hybridization on microtiter plates. We tested 188 individuals recruited from the genetic counseling service for deaf people at the Pasteur Hospital and at the Armand-Trousseau Children's Hospital, Paris, France between April 1997 and September 1998. Our screening method is simple, uses stable and safe reagents, and employs inexpensive equipment. As such, it is suitable for widespread use in genetic diagnosis.

Evaluation of IS6110 as amplification target for direct tuberculosis diagnosis.

We describe in the present study an evaluation of the IS6110 repetitive element in the rapid diagnosis of pulmonary and extrapulmonary tuberculosis by polymerase chain reaction (PCR). A pair of oligonucleotide primers was designed to amplify a 201-bp DNA fragment of IS6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by Sal I digestion and Southern blot hybridization with a 32P-labeled probe. To detect the presence of amplification inhibitors, an internal control DNA that used the same primers as for the target sequence was added to each PCR reaction. PCR results were compared with the results of acid fast stained smears, cultures, and clinical data in 102 sputum and 41 extrapulmonary specimens. With the exception of four samples, M. tuberculosis was detected by PCR in all smear- and culture-positive cases and in all smear-negative, culture positive cases. Additionally, PCR was able to detect 6 cases that were smear and culture negative but clinically strongly suspected of tuberculosis. The final PCR sensitivity and specificity were 93.1% and 95.18%, respectively. One M. tuberculosis strain isolated from a sputum was found to lack IS6110. This study shows that (1) PCR diagnosis based on IS6110 reached the best sensitivity and specificity but must be considered carefully since some M. tuberculosis strains lack IS6110; and (2) PCR must be interpreted in conjunction with clinical and radiological data when it is discordant with conventional methods results.

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization.

Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

Evaluation of an rDNA Listeria probe for Listeria monocytogenes typing.

A Listeria monocytogenes DNA fragment, identified as part of the 23S rRNA gene and called B17, was used to type 266 L. monocytogenes strains and 43 strains of other Listeria species. Results were compared with those obtained: i) with pBA2 (which consists of a 2.3 kb Bacillus subtilis DNA fragment encoding 16S rRNA, inserted into the HindIII site of pBR322), a probe previously used for Listeria and L. monocytogenes ribotyping, and ii) with DNA macrorestriction profiles analysis. Twenty profiles were identified for L. monocytogenes using pB 17, three of which accounted for 87% of strains. This new rDNA probe had greater discriminatory power for serogroups 1/2 or 3 strains than for serogroup 4 strains. The number of varieties and the discrimination index were higher with this new probe than with pBA2, but DNA macrorestriction patterns analysis gave better discrimination between strains.

Isolation of a specific DNA fragment and development of a PCR-based method for the detection of Mycobacterium genavense.

The rise of Mycobacterium genavense infections is making identification ever more important for diagnosis and treatment. Moreover, isolation and identification of M. genavense are made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplification by PCR or similar techniques represents the only possibility of detecting and identifying M. genavense from tissue samples. In order to set up a simple and species-specific method based on the use of PCR and non-radioactive hybridization technique, we decided to search for and clone a specific DNA fragment of this bacterial species. In the present study, a 1734-bp fragment was isolated. This fragment was found to be highly specific for M. genavense strains. A species-specific pair of primers (MG22 and MG23) and two oligonucleotide probes (MG18 and MG19) were selected. They were successfully used to amplify and detect a 155-bp DNA fragment from the 13 available strains of M. genavense which were isolated from clinical specimens or from birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, although it is closely related to M. genavense. The present PCR technique uses species-specific primers for M. genavense. Followed by a non-radioactive hybridization technique on microplates it is able to distinguish M. genavense from other mycobacteria in one step, without sequencing or restriction analysis. On the basis of the Southern blot hybridization, PCR and sandwich hybridization results, we concluded that the isolated 1.7-kb sequence was specific for the M. genavense chromosome. The method developed here for M. genavense identification uses a simple methodology and commonly available reagents. Furthermore it can be easily automated.

Cloning of a sapB homologue (sapB2) encoding a putative 112-kDa Campylobacter fetus S-layer protein and its use for identification and molecular genotyping.

A sap gene encoding a surface layer protein was isolated from a Campylobacter fetus ssp. fetus CIP 53.96T cosmid library. This sap gene, which shows significant homology with the sapB conserved region, was named sapB2. The complete ORF of 3339 nucleotides encodes a 1112-amino acid polypeptide with a calculated molecular mass of 112 kDa. High homology with the sapB gene was found in a region beginning 67 bp before the ORF and proceeding 546 bp into the ORF. Similarly, 98% homology with the sapA2 gene was observed in a 2038-bp region beginning 540 bp after the initiation codon. In the present study, we show that this sapB2 gene has two main interesting features: the 5' end of the region which presents high homology with the sapA2 homologue was found to be present in every C. fetus strain, and the fragment (IG01) comprising the region which presents homology with the sapB conserved region and the 5' end of the sapA2 homologue region, when used as a probe, can reveal genomic polymorphism among C. fetus strains. We exploited these features to develop a PCR assay for the specific detection of C. fetus and to set up a method for typing C. fetus isolates. The PCR assay was found to be species-specific. Oligonucleotide primers derived from the 5' end of sapA2 homologue region were used in a polymerase chain reaction test on genomic DNA extracted from 101 Campylobacter fetus, 18 Campylobacter non-fetus and seven non-Campylobacter strains. A 220-bp fragment was amplified only when C. fetus DNA was used as a target. In Southern blot analysis, the IG01 probe was found to hybridize only with DNA extracted from C. fetus strains. Moreover, IG01 hybridized with several fragments of HindIII-digested DNA, giving a specific pattern for each strain.

Phage-displayed and soluble mouse scFv fragments neutralize rabies virus.

A phage-display technology was used to produce a single-chain Fv antibody fragment (scFv) from the 30AA5 hybridoma secreting anti-glycoprotein monoclonal antibody (MAb) that neutralizes rabies virus. ScFv was constructed and then cloned for expression as a protein fusion with the g3p minor coat protein of filamentous phage. The display of antibody fragment on the phage surface allows its selection by affinity using an enzyme-linked immunosorbent assay (ELISA); the selected scFv fragment was produced in a soluble form secreted by E. coli. The DNA fragment was sequenced to define the germline gene family and the amino-acid subgroups of the heavy (VH) and light (VL) chain variable regions. The specificity characteristics and neutralization capacity of phage-displayed and soluble scFv fragments were found to be identical to those of the parental 30AA5 MAb directed against antigenic site II of rabies glycoprotein. Phage-display technology allows the production of new antibody molecule forms able to neutralize the rabies virus specifically. The next step could be to engineer and produce multivalent and multispecific neutralizing antibody fragments. A cocktail of multispecific neutralizing antibodies could contain monovalent, bivalent or tetravalent scFv fragments, for passive immunoglobulin therapy.

Acetoxy-acetylaminofluorene-modified dGTP can be used to label oligonucleotides or DNA enzymatically.

A competitive enzyme immunoassay using a bispecific monoclonal antibody (Bi-MAb) was developed to quantify acetylaminofluorene (AAF) adducts fixed on DNA and then compared to the spectrophotometric method. It was shown that this simple method allowed the measurement of as low as 2 pmol per assay of AAF bound to DNA. This technique was used to monitor synthesis and purification of N-acetoxy-N-2-acetylaminofluorene modified dGTP (AAF-dGTP). It was shown that AAF-dGTP can be a substrate for the terminal deoxynucleotidyl transferase. Finally, using the Bi-MAb we developed a non-radioactive sandwich hybridization assay making use of oligonucleotide covalently bound to microwells and of synthetic oligonucleotide tailed with AAF-dGTP as a probe.

Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA.

Bacillus anthracis can be identified on the basis of the detection of virulence factor genes located on two plasmids, pXO1 and pXO2. Thus isolates lacking both pXO1 and pXO2 are indistinguishable from closely related B. cereus group bacteria. We developed a multiplex PCR assay for characterization of B. anthracis isolates, and simultaneous confirmation of the species identity independent of plasmid content. The assay amplifies lef, cya, pag (pXO1) and cap (pXO2) genes, and a B. anthracis specific chromosomal marker, giving an easy-to-read profile. This system unambiguously identified virulent (pXO1+/2+) and avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished "anthrax-like' strains from other B. cereus group bacteria.

Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis.

A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis.

Characterization of the type strain of Campylobacter coli, CIP 70.80, by plasmid typing.

A 1.9-kb plasmid DNA fragment from the type strain of Campylobacter coli, CIP 70.80, was used as a probe to characterize this type strain, other C. coli type strains obtained from several culture collections, and other C. coli strains. A specific hybridization pattern was obtained, and this pattern can be used to identify, characterize, and follow up C. coli type strains in culture collections.

Rapid detection of Campylobacter jejuni and Campylobacter coli isolated from clinical specimens using the polymerase chain reaction.

Seventeen Campylobacter strains isolated from 16 children hospitalised with acute diarrhea were analysed by in vitro enzymatic amplification using two sets of oligonucleotide primers specific for Campylobacter jejuni and Campylobacter coli, respectively. Thirteen strains (76%) were identified as Campylobacter jejuni and four strains (24%) as Campylobacter coli. Subsequent bacteriological identification confirmed the identity of the same 13 Campylobacter jejuni strains and the 4 Campylobacter coli strains. Thus, these PCR methods enabled rapid and specific detection of all the Campylobacter jejuni and Campylobacter coli strains without any false-positive or false-negative results.

Characterization of serogroup A Neisseria meningitidis strains by rRNA gene restriction patterns and PCR: correlation with the results of serotyping, subtyping and multilocus enzyme electrophoresis.

We studied 35 strains of Neisseria meningitidis serogroup A from different locations (France, Central African Republic, Sudan and Burkina Faso) using both ribotyping and a polymerase chain reaction (PCR). A non-radioactive probe label was used for ribotyping; detection consisted of an immunoenzymatic procedure using a bispecific antibody. The PCR was designed to amplify the 16S-23S rDNA internal transcribed spacer. These techniques were compared with other markers. The strains were identified as belonging to three clones (I, III-1, IV) by multilocus enzyme electrophoresis (MEE) and to three subtypes by serological methods. Ribotyping identified five groups and PCR identified four groups. Ribotyping gave more diversity between strains than either MEE or sero/subtyping, but confirmed the epidemiological data provided by the combination of these two techniques. The PCR provided a simple and convenient one-step procedure for the differentiation of strains of serogroup A.