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PubMed2004

Multiplex opsonophagocytosis assay (MOPA): a useful tool for the monitoring of the 7-valent pneumococcal conjugate vaccine.

Pneumococcal conjugate vaccination is highly efficacious against invasive diseases in young children. Since host protection is mainly mediated by opsonin-dependent phagocytosis, the in vitro measurement of opsonophagocytic activity of the anti-capsular antibodies is assumed to be a reliable correlate of protection to monitor vaccine efficacy. Unfortunately, the methods used so far are all tedious to perform and material-consuming. Therefore, we modified the multi-specificity opsonophagocytosis killing assay (MSOPKA) into a high-throughput method, which simultaneously measures the opsonophagocytosis against the seven serotypes covered by the current conjugate vaccine in a single assay. In the so-called multiplex opsonophagocytosis assay (MOPA), a mixture containing equal numbers of colony forming units (CFUs) of chloramphenicol-resistant serotype 4, spectinomycin-resistant serotype 6B, streptomycin-resistant serotype 9V, erythromycin-resistant serotype 14, rifampicin-resistant serotype 18C, tetracycline-resistant serotype 19F, and trimethoprim-resistant serotype 23F pneumococci was used as a target mixture and incubated with serial dilutions of test serum. After opsonophagocytosis by differentiated HL-60 cells in the presence of complement, the samples were spotted onto different blood agar plates containing the seven selective antibiotics, respectively. Opsonophagocytosis was calculated as the highest serum dilution resulting in 90% or more reduction in CFUs. The data obtained by this assay correlated well with the data obtained by the MSOPKA. In conclusion, the MOPA simultaneously measures opsonophagocytosis capacity of serum against the capsular serotypes included in the 7-valent pneumococcal conjugate vaccine in a high-throughput fashion, requiring low volumes of patient sera.

Antibodies, Bacterial↗
PubMed2005

Modified multiplex PCR for identification of Bacillus Calmette-Guérin substrain Tokyo among clinical isolates.

When an adverse reaction occurs and a mycobacterial species is isolated from a person vaccinated with Bacillus Calmette-Guérin (BCG) or a patient receiving BCG immunotherapy, it is essential to identify whether the isolate is BCG or another mycobacterial species. However, differentiation of BCG from other members of Mycobacterium tuberculosis complex has been very difficult. Using several specific primer-pairs, Bedwell et al. [Bedwell J, Kairo SK, Behr MA, Bygraves JA. Identification of substrains of BCG vaccine using multiplex PCR. Vaccine 2001; 19: 2146-51] recently reported that they could distinguish BCG substrains. We modified their method to improve differentiation of Tokyo 172 from other members of the M. tuberculosis complex, and examined whether this modified method could be applied to clinical isolates. Our method clearly identified BCG substrain (BCG Tokyo 172) among clinical isolates and easily distinguished between M. tuberculosis and wild-type Mycobacterium bovis.

Adjuvants, Immunologic↗
PubMed2004

Multiplex PCR for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses.

A multiplex PCR (mPCR) assay was developed and subsequently evaluated for its effectiveness as a means to simultaneously detect multiple viral infections of swine. Specific primers for each of four common DNA viruses, namely, pseudorabies virus (PRV), porcine circovirus type I (PCV1), porcine circovirus type II (PCV2), and porcine parvovirus (PPV), were used for testing procedure. The assay was shown to be highly sensitive in that as little as 10(-4) ng of each of the respective amplicons (approximately equal to 10,000 molecules) was detected when a composite of all four viruses (including both field and gene-deleted permutations of PRV) was tested as a single sample. It was also effective for detecting one or more of these same viruses in various combinations in specimens including lymph nodes, lungs, spleens, and tonsils collected from clinically ill pigs, and in specimens in spleen collected from aborted fetuses. The relative efficiency (compared to performing separate assays for each virus) and apparent sensitivity of mPCR suggest its potential application for routine molecular diagnostic purposes.

Animals↗
PubMed2005

Differentiation of five strains of infectious bursal disease virus: development of a strain-specific multiplex PCR.

Infectious bursal disease virus (IBDV) is a major cause of disease problems in the poultry industry and vaccination has therefore been applied intensively to control the infection. The classical methods of detection and characterization of IBDV are by the use of immunodiffusion test and histopathology. Since these methods are laborious and have low specificity alternatives are needed. In the present study, we report the development of a strain-specific multiplex RT-PCR technique, which can detect and differentiate between field strains of IBDV and vaccine virus strains including a so-called hot vaccine strain widely used in the European poultry industry. The method, which is highly specific, fast and inexpensive, can be applied in all laboratories with basal PCR capabilities and equipment.

Animals↗
PubMed2005

Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR.

The aim of this work was the design and validation of a rapid and easy single tube multiplex-PCR (m-PCR) assay for the unequivocal differential detection of Mycobacterium bovis and Mycobacterium tuberculosis. Oligonucleotide primers were based on the uninterrupted 229-bp sequence in the M. bovis genome and a unique 12.7-kb insertion sequence from the M. tuberculosis genome, which is responsible for species-specific genomic polymorphism between these two closely related pathogens. The m-PCR assay was optimized and validated using 22 M. bovis and 36 M. tuberculosis clinical strains isolated from diverse host species and 9 other non-tuberculous mycobacterial (NTM) strains. The designed primers invariably amplified a unique 168-bp (M. bovis-specific) and 337-bp (M. tuberculosis-specific) amplicon from M. bovis and M. tuberculosis strains, respectively. The accuracy of the assay, in terms of specificity, was 100%, as none of the NTM strains tested revealed any amplification product. As little as 20 pg of genomic DNA could be detected, justifying the sensitivity of the method. The m-PCR assay is an extremely useful, simple, reliable and rapid method for routine differential identification of cultures of M. bovis and M. tuberculosis. This m-PCR may be a valuable diagnostic tool in areas of endemicity, where bovine and human tuberculosis coexist, and the distinction of M. bovis from M. tuberculosis is required for monitoring the spread of M. bovis to humans.

Animals↗
PubMed2006

Development of a multiplex PCR assay for polymorphism analysis of Brucella suis biovars causing brucellosis in swine.

Swine brucellosis is caused by the biovars 1, 2 and 3 of Brucella suis the identification of which up to now relies on microbiological tests lacking adequate specificity together with time consuming and expensive molecular procedures. Based on sequence variation of the omp2b gene, we have developed a four primer set multiplex PCR assay that was tested for polymorphism analysis of B. suis biovars causing brucellosis in swine. The assay exploits the single nucleotide polymorphisms found in omp2b gene of B. suis reference biovars which are conserved in 43 B. suis field isolates from different geographic origins and hosts. Three specific amplification patterns (S1, S2 and S3) were obtained for reference strains of B. suis biovars 1, 2 and 3, respectively. However, some B. suis field isolates identified as biovars 2 or 3 according AMOS-PCR, PCR-RFLP of omp31 and omp2 genes and classical bacteriological methods, resulted also in S1 patterns, limiting the typing usefulness of the method.

Amino Acid Sequence↗
PubMed2006

A one-step multiplex real-time RT-PCR for detection and typing of bovine viral diarrhea viruses.

A one-step multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using SmartCycler technology and TaqMan probes was developed for detection and typing of bovine viral diarrhea viruses (BVDV). Common primers and type-specific (BVDV1 and BVDV2) TaqMan probes were designed in the 5'-untranslated region of the viral genome. The real-time assay was able to detect 10-100 TCID50 of virus, with correlation coefficient (r2) values of 0.998 and 0.999 for BVDV1 and BVDV2, respectively. The assay accurately typed 54 BVDV strains and field isolates and specificity of the TaqMan probes was further demonstrated by the lack of reactivity with the closely related Pestiviruses, classical swine fever virus and border disease virus. The assay was also shown to have high reproducibility. When the assay was compared with virus isolation for bovine serum samples, there was full agreement between the tests. Thus, the one-step real-time RT-PCR assay appears to be a rapid, sensitive, and specific test for detection and typing of BVDV.

Animals↗
PubMed2006

Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR.

An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.

Animals↗
PubMed2006

Multiplex locked nucleic acid probes for analysis of hepatitis B virus mutants using real-time PCR.

Current methods of detecting hepatitis B virus (HBV) mutations are time consuming, labor intensive, and not suitable for screening large numbers of samples. A multiplex real-time PCR approach presented in this article is a hepatitis B virus quantification method that employs the SYBR Green I dye in conjunction with wild-type HBV sequence-specific locked nucleic acid (LNA) probes. The three short LNA probes distinguished the wild-type strain or three groups of mutants (rt173, rt180/rt181, and rt202/rt204) depending on perfect-match hybrids or mismatch within one template simultaneously. Primers labeled with quencher minimized the background signals. This sensitive approach could quantify 10(2) copies of HBV virus, and as low as 1% mutants among 10(4) copies of wild-type HBV could be identified. The technique is handy and convenient, requiring only 3.5 h to analyze 30 hepatitis B surface antigen-positive serum samples. The HBV isolates were confirmed by direct sequencing. Our data indicate that real-time PCR with SYBR Green I dye is a reliable, rapid, and convenient technique for HBV quantification. Furthermore, by incorporating fluorescent LNA probes, this technique becomes handy in identifying and classifying mutations in the HBV polymerase gene. Being sensitive, specific, accurate, rapid, and convenient in nature, this technique could be a suitable diagnostic tool with wide application particularly in cases in which large volumes of clinical samples are handled.

Base Sequence↗
PubMed2005

Short order nanohole arrays in metals for highly sensitive probing of local indices of refraction as the basis for a highly multiplexed biosensor technology.

A small array of subwavelength apertures patterned in a gold film on glass was characterized for use as a biosensor. It is widely believed that such arrays allow the resonance of photons with surface plasmons in the metallic film. Surface plasmon methods (and other evanescent wave methods) are extremely well suited for the measure of real time biospecific interactions. An extremely high sensitivity of 88,000%/refractive index unit was measured on an array with theoretical active area of .09 microm2. The formation of a biological monolayer was monitored. Both sensitivity and resolution were determined through measurement. The measured resolution, for a sensor with an active area of less than 1.5 microm2, is 9.4 x 10(-8) refractive index units which leads to a calculated sensitivity of 3.45E6%/refractive index unit. These values far exceed theoretical and calculated values of other grating coupled surface plasmon resonance (SPR) detectors and prism based SPR detectors. Because the active sensing area can be quite small (.025 microm2) single molecule studies are possible as well as massive multiplexing on a single chip format.

Biosensing Techniques↗
PubMed2004

Defining a 0.5-mb region of genomic gain on chromosome 6p22 in bladder cancer by quantitative-multiplex polymerase chain reaction.

Metaphase-based comparative genomic hybridization (CGH) has identified recurrent regions of gain on different chromosomes in bladder cancer, including 6p22. These regions may contain activated oncogenes important in disease progression. Using quantitative multiplex polymerase chain reaction (QM-PCR) to study DNA from 59 bladder tumors, we precisely mapped the focal region of genomic gain on 6p22. The marker STS-X64229 had copy number increases in 38 of 59 (64%) tumors and the flanking markers, RH122450 and A009N14, had copy number gains in 33 of 59 (56%) and 26 of 59 (45%) respectively. Contiguous gain was present for all three markers in 14 of 59 (24%) and for two (RH122450 and STS-X64229) in 25 of 59 (42%). The genomic distance between the markers flanking STS-X64229 is 0.5 megabases, defining the minimal region of gain on 6p22. Locus-specific interphase fluorescence in situ hybridization confirmed the increased copy numbers detected by QM-PCR. Current human genomic mapping data indicates that an oncogene, DEK, is centrally placed within this minimal region. Our findings demonstrate the power of QM-PCR to narrow the regions identified by CGH to facilitate identifying specific candidate oncogenes. This also represents the first study identifying DNA copy number increases for DEK in bladder cancer.

Adult↗
PubMed1999

Multiplex Western blotting system for the analysis of muscular dystrophy proteins.

A multiplex system of Western blotting is presented in which most of the current muscular dystrophy proteins can be analyzed simultaneously on one pair of blots. This represents a significant improvement in efficiency and cost for this type of analysis. The final diagnosis is more quickly achieved in patients where several possible diagnoses are indicated after clinical appraisal, and those with unusual presentations may be quickly resolved. The method uses a biphasic polyacrylamide gel system, which enables the corresponding blot to be probed simultaneously with a cocktail of monoclonal antibodies. The gel is optimized so that large proteins of more than 200 kd (eg, dystrophin, dysferlin, and myosin heavy chain) can be analyzed in the top part, while smaller proteins under 150 kd (eg, calpain 3, the 80-kd fragment of laminin alpha2 chain, all of the sarcoglycans, and caveolin 3) are separated in the lower phase. This basic system could be used for different combinations of antibodies as new muscular dystrophy proteins are identified and require examination. In addition, analysis of the laminin alpha2 chain of merosin showed that this protein was expressed as a doublet or triplet set of bands in many patients with active muscle pathology. This may indicate the existence of an embryonic isoform, which is re-expressed in regenerating fibers.

Biopsy↗
PubMed2001

Simultaneous amplification of DNA and RNA virus using multiplex PCR system.

A large number of disease-causing bacteria and viruses are being sequenced and PCR is increasingly used for the diagnosis of the diseases. We have designed a multiplex PCR system for hepatitis B virus (HBV), a DNA virus, and hepatitis E virus (HEV), an RNA virus. A modified technique has been standardized for simultaneous extraction of DNA and RNA, followed by a one-step RT-PCR/PCR.

DNA↗
PubMed2003

Analysis of the Glutathione S-transferase M1 gene using pyrosequencing and multiplex PCR--no evidence of association to glaucoma.

The Glutathione S-transferase M1 (GSTM1) gene is reported to be involved in glaucoma, an eye disease with a largely unknown mechanism. The gene is polymorphic and three alleles have been characterized. These are one complete deletion of the gene, GSTM1*0, and two alleles differing only in a single amino acid substitution, GSTM1*A and *B. The two latter alleles seem to have equivalent function. Approximately 45% of the European populations are GSTM1 positive. An Estonian study has found that 60% of the glaucoma patients are GSTM1 positive as compared to 45% of controls (P=0.002). We genotyped 200 primary open angle glaucoma patients (POAG), 188 exfoliative glaucoma patients and 200 matched controls using multiplex PCR and pyrosequencing. Forty four per cent of the POAG patients and exfoliative glaucoma patients, and 44.5% of the matched controls were GSTM1 positive. Using pyrosequencing we were able to determine if the patients were homo- or hemizygous for the GSTM1 gene. Five per cent of the POAG patients, 7.4% of the exfoliative glaucoma patients and 4.6% of the controls were homozygous for the presence of the GSTM1 gene. There is no evidence of association between GSTM1 and glaucoma in the Swedish population.

Base Sequence↗
PubMed1999

The identification of Cryptosporidium species and Cryptosporidium parvum directly from whole faeces by analysis of a multiplex PCR of the 18S rRNA gene and by PCR/RFLP of the Cryptosporidium outer wall protein (COWP) gene.

A multiplex polymerase chain reaction (PCR) procedure to amplify 18S rRNA gene fragments has been developed. Amplified DNA fragments of the expected size were obtained which were specific for Cryptosporidium parvum and Cryptosporidium wrairi (422 bp), Cryptosporidium baileyi (11106 bp) and Cryptosporidium muris (1346 bp). Criptosporidium parvum and C. wrairi can be distinguished using a PCR/restriction fragment length polymorphism (RFLP) analysis of the Cryptosporidium outer wall protein (COWP) gene, and these two techniques were applied to DNA extracted from whole faeces using a simple and rapid procedure. Cryptosporidium parvum DNA was detected in the faeces of 72 humans and 24 calves where cryptosporidial oocysts were demonstrated using conventional light microscopy. The specific DNA fragments were not amplified using extracts of material containing other lower eukaryotic parasites.

Animals↗
PubMed2001

A multiplex PCR-based DNA assay for the detection of paraoxonase gene cluster polymorphisms.

Paraoxonase (PON) is a high-density lipoprotein (HDL) associated protein which is supposed to protect low-density lipoprotein (LDL) against oxidation and to play a role in the development of atherosclerosis. Interindividual variability in serum PON activity is attributable to common variants in components of the PON gene cluster on chromosome 7. We describe experimental conditions that permit the simultaneous determination of three common PON polymorphisms (PON1-192, PON1-55 and PON2-311) that are tightly associated with an increased risk of atherosclerosis. We used a multiplex PCR-based DNA assay using mismatch primers that introduce a unique recognition site for the endonuclease HinfI in the PCR products in case of presence of the R allele of PON 1-192, of the L allele of PON1-55 and of the S allele of PON2-311. The restriction analysis with HinfI allows to identify an electrophoretic band pattern which is specific for the combination of the three polymorphisms. This technique could be applied in the association studies aimed at assessing the role of PON and their polymorphisms in many clinical settings. In a preliminary study on a small population sample from south Italy about 10% of chromosomes exhibited the presumed risk-related haplotype R(192)/L(55)/S(311).

Alleles↗
PubMed2002

Multiplexed capillary electrophoresis for DNA sequencing with ultra violet absorption detection.

DNA sequencing is performed in a multiplexed capillary electrophoresis system by UV absorption detection. Four individual electropherograms are obtained by simultaneously running the unlabeled DNA products of the four ddNTP-terminated reactions in the capillary array. The sequence of the template used in the cycle-sequencing reaction can be determined by overlaying the four electropherograms. Two internal standards are employed to adjust for the variance in migration times among the capillaries. After applying the correction algorithm, base calling can be done at a high level of confidence.

Electrophoresis, Capillary↗
PubMed2001

Multiplex screening for functionally rearranged immunoglobulin variable regions reveals expression of hybridoma-specific aberrant V-genes.

Modification of antibody effector functions is commonly performed by chimerization or humanization. Cloning of antibody variable regions from hybridomas represents a first step that is frequently hampered by the expression of non-functionally rearranged variable regions in hybridoma cells that originate from MOPC21-derived fusion partners. We now present a simple method to clone functionally rearranged V-genes, based on V-gene-specific multiplex PCR screening. Using this method we document the expression of aberrant V-genes that originate from the original B-cell used for the hybridoma generation, not from the fusion partner, and are - thus - hybridoma specific.

Animals↗