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Multiplex detection of RNA expression in Drosophila embryos.

We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven differ-ent transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent transcript multiplex fluorescent in situ hybridization. These multiplex methods fill a current technological gap between high-resolution in situ hybridization with one or two fluorescently labeled probes and low-resolution but genome-wide microarray RNA profiling and should be of great utility in establishing gene networks.

Animals↗

Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates.

A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of 138 clinical isolates of Mycobacterium tuberculosis. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the gyrA gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained gyrA mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains </= 4 mg/liter) were apparently more active than ofloxacin, levofloxacin, and sparfloxacin (MIC for 90% of strains >/==" BORDER="0"> 16 mg/liter). All gyrA mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without gyrA mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains = 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains </= 1 mg/liter). Another 83 isolates without mutations were fully susceptible to all six fluoroquinolones (ofloxacin MIC for 90% of strains = 1 mg/liter). In conclusion, high-level phenotypic resistance to fluoroquinolones among M. tuberculosis clinical isolates, which appears to be predominantly due to gyrA mutations, may be readily detected by genotyping techniques such as multiplex PCR amplimer conformation.

Anti-Infective Agents↗

New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates.

In this study, we describe a multiplex PCR to detect a AGC-->ACC (serine to threonine) mutation in the katG gene and a -15 C-to-T substitution (inhA(C-15T)) at the 5' end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.

Antitubercular Agents↗

Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community.

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.

Archaea↗

Multiplex PCR assay for detection of bacterial pathogens associated with warm-water Streptococcosis in fish.

A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

Animals↗

Heterophile antibody interference in a multiplexed fluorescent microsphere immunoassay for quantitation of cytokines in human serum.

While modern immunoassays provide sensitive and specific means for the quantitation of cytokines in biological fluids, heterophile antibodies are still a well-recognized cause of interference in the measurement of cytokines in these assays. We have developed a multiplexed fluorescent microsphere immunoassay for the simultaneous quantification of 10 cytokines in only 75 microl of serum. During the development of this multiplexed assay, the amount of assay interference due to heterophile antibodies was also determined, and methods for detecting heterophile interference and minimizing its effect were incorporated into the assay. Heterophile antibodies resulted in significantly elevated cytokine values compared to those of normal blood bank samples. These falsely elevated values, and thus the components of the assay the heterophile antibodies were binding to, were identified through the use of internal controls. This information was then used to design assay-specific blockers and absorbents that were shown to significantly reduce falsely elevated cytokine values while not affecting the standard and control values. The fluorescent multiplexed microsphere-based immunoassay can be used to quantitate multiple cytokines from a single sample and should be a useful tool in furthering our understanding of the role of cytokines in disease processes.

Antibodies, Heterophile↗

Simultaneous detection of antibodies to six nonhuman-primate viruses by multiplex microbead immunoassay.

To maintain healthy nonhuman primates for use in biomedical research, animals are routinely screened for several infectious agents at most facilities. Commonly, monkey serum samples are tested by conventional immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, for antibodies to specific infectious agents. For testing for antibodies against multiple agents in each sample, conventional immunoassays are laborious and time-consuming. More efficient immunoassays are needed. Accordingly, we have developed a novel multiplex serodiagnostic system based on individually identifiable, fluorescent microbead sets, where each bead set is coated with antigens from a purified preparation of a specific virus. The coated bead sets are mixed to enable the detection of antibodies to multiple viruses in one serum or plasma sample. These viruses include four agents that are routinely tested for maintenance of specific-pathogen-free monkeys, namely, simian immunodeficiency virus, simian type D retrovirus, simian T-cell lymphotropic virus, and herpes B virus, as well as simian foamy virus and rhesus cytomegalovirus, both of which are commonly found in nonhuman primates. This multiplex microbead immunoassay (MMIA) enabled the simultaneous detection of antibodies to all six viruses in single serum samples as small as 1 microliter. The results obtained by MMIA analysis correlated with results of conventional ELISAs, which detect antibodies to single agents. Thus, this multiplex microbead detection system is an efficient diagnostic modality for serosurveillance of nonhuman primates.

Animals↗

Multiplex real-time PCR targeting the RNase P RNA gene for detection and identification of Candida species in blood.

We have developed a single-tube multiplex real-time PCR method for the detection of the eight most common Candida species causing septicemia: Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. tropicalis. The method developed targets the RNase P RNA gene RPR1. Sequences of this gene were determined for seven of the Candida species and showed surprisingly large sequence variation. C. glabrata was found to have a gene that was five times longer gene than those of the other species, and the nucleotide sequence similarity between C. krusei and C. albicans was as low as 55%. The multiplex PCR contained three probes that enabled the specific detection of C. albicans, C. glabrata, and C. krusei and a fourth probe that allowed the general detection of the remaining species. The method was able to detect 1 to 10 genome copies when the detection limit was tested repeatedly for the four species C. albicans, C. glabrata, C. krusei, and C. guilliermondii. No significant difference in the detection limit was seen when the multiplex format was compared with single-species PCR, i.e., two primers and one probe. The method detected eight clinically relevant Candida species and did not react with other tested non-Candida species or human DNA. The assay was applied to 20 blood samples from nine patients and showed a sensitivity similar to that of culture.

Base Sequence↗

Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types.

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.

DNA Primers↗

Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens.

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.

Actins↗

Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans and P. gingivalis cells. Primer specificity was tested against (i) six A. actinomycetemcomitans strains and four P. gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects. The multiplex PCR had a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. gingivalis cells. Species-specific amplicons were obtained for all A. actinomycetemcomitans and P. gingivalis strains tested and did not occur with seven other bacterial species unless A. actinomycetemcomitans and P. gingivalis were added. Neither target species was detected in supragingival plaque; A. actinomycetemcomitans was detected in one subgingival specimen, and P. gingivalis was detected in another. When plaque samples were spiked with 10 A. actinomycetemcomitans cells and 100 P. gingivalis cells, species-specific amplicons were detected. These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A. actinomycetemcomitans and P. gingivalis. This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens.

Aggregatibacter actinomycetemcomitans↗

Multiplex PCR provides a low-cost alternative to DNA probe methods for rapid identification of Mycobacterium avium and Mycobacterium intracellulare.

A multiplex PCR designed to differentiate Mycobacterium tuberculosis complex organisms from M. avium and M. intracellulare was used to test 105 isolates identified by DNA probe methods as M. avium, M. intracellulare, or M. avium complex type X. The multiple PCR correctly identified 33 of 34 isolates identified by commercial probe methods as M. avium and all 51 isolates identified as M. intracellulare. The 20 isolates identified as M. avium complex type X by probe were identified as Mycobacterium spp. by the multiplex method. These results confirm that the multiplex PCR, which is simple to perform and cheaper than commercial probe methods, is suitable for routine identification of M. avium and M. intracellulare.

Costs and Cost Analysis↗

Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions.

A multiplex PCR procedure was developed for the simultaneous detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae in middle ear effusions (MEEs) from patients with chronic otitis media with effusion. The bacterial 16S rRNA gene was chosen as the target, and the procedure used one common lower primer and four species-specific upper primers. The reaction was optimized by changing the primer concentrations to yield equal amounts of amplification products. The specificity of the reaction was verified with various bacterial species found in the nasopharynx. The performance of the procedure was examined with 25 MEE specimens, and the results were compared to those obtained by conventional culture methods. A detection level of 10 bacterial cells/reaction for each of the study organisms was achieved. By conventional culture methods, 8 (32%) of the specimens showed growth of one of the study organisms. In contrast, 21 (84%) of the specimens tested positive by the multiplex PCR. None of the culture-positive specimens were PCR negative, whereas three (12%) of the PCR-positive specimens tested positive for two of the four study organisms. Thus, the multiplex PCR method improves the detection rate significantly compared to that of the conventional culture method.

Child↗

Direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-PCR assay.

Diagnosis of respiratory virus infections currently involves detection by isolation or antigen detection, which usually identifies only a single suspected agent. To permit identification of more than one respiratory virus in clinical specimens, a rapid detection method involving a single-step, multiplex reverse transcription-PCR (RT-PCR) assay was developed. The assay included five primer sets that amplified the RNA of respiratory syncytial virus subtypes A and B, parainfluenza virus types 1, 2, and 3, and adenovirus types 1 to 7. Initially the assay was tested on tissue culture-grown virus and was found to be specific for all 12 prototype viruses tested, with no interassay cross amplification or amplification of other respiratory viruses. Assay sensitivity allowed a detection range of 0.2 50% tissue culture infectious dose (TCID50) for adenovirus to 250 TCID50 for parainfluenza virus type 1. The multiplex RT-PCR assay was also able to directly detect viruses in respiratory specimens, with virus being detected in 41 of 112 samples as compared to 34 of 112 samples detected by direct immunofluorescence or antigen detection following specimen culture. This suggests that the multiplex RT-PCR assay can be used as a rapid and sensitive diagnostic method for major respiratory viruses.

Adenovirus Infections, Human↗

Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene.

Two multiplex PCR enzyme immunoassays (PCR-EIAs) were developed for Staphylococcus aureus exotoxin gene screening as an alternative to the conventional biological assays, which depend on detectable amounts of toxins produced. One set of oligonucleotide primers and probes was designed to search for enterotoxin A to E genes (entA, entB, entC, entD, and entE), and the other one was designed to detect the staphylococcal exfoliative toxin genes (eta and etb) and the toxic shock syndrome toxin 1 gene (tst). Oligonucleotide primers were used as published previously, modified or newly developed to meet the requirements of both good size-distinguishable amplification bands of multiplex PCR and the temperature limit of the uracil DNA glycosylase system for carryover protection. Amplification products were visualized by agarose gel electrophoresis, and specificity was controlled with the aid of a DNA EIA system using oligonucleotide probes derived from the sequences of the S. aureus toxin genes. PCR procedures were performed by using template nucleic acids extracted from a panel of S. aureus reference strains and from a collection of 50 clinical strains. The PCR results were compared with those of immunological toxin production assays. This multiplex PCR-EIA system offers an alternative method for the rapid, sensitive, specific, and simultaneous detection of the clinically important exotoxin potency of isolated S. aureus strains for diagnostic purposes as well as research studies.

Bacterial Toxins↗

Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers.

Integration of human papillomavirus (HPV) DNA occurs early in cancer development and is an important event in malignant transformation of cervical cancer. Integration of HPVs preferentially disrupts or deletes the E2 open reading frame, which results in the loss of its expression. The preferential disruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA were first tested for the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, suggesting integration of HPV DNA into the host genome. Next, multiplex PCR for the HPV E2 and E6 genes was carried out in the remaining 12 cases. Copy numbers of both genes should be equivalent in episomal forms, while the E2 gene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concomitant forms. Although relative ratios of HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 of 12 cases, multiplex PCR products from 2 cases displayed extremely low ratios of 0.69 and 0.61. Southern blot hybridization with an HPV-16 probe revealed that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes of HPV-16 DNA following PCR for the E2 gene can distinguish the pure episomal form from a mixed form of episomal and integrated HPV DNA. Clinical application of this technique will help researchers to understand the implication of the integration of HPV DNA for cervical carcinogenesis and cervical cancer progression.

Base Sequence↗

Simultaneous detection of multiplex-amplified human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay.

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from the gag region of the HIV-1 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from the 5' noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.

Acquired Immunodeficiency Syndrome↗

Diagnostic utility of a multiplex herpesvirus PCR assay performed with cerebrospinal fluid from human immunodeficiency virus-infected patients with neurological disorders.

We used a multiplex nested-PCR assay for the simultaneous detection in cerebrospinal fluid (CSF) of five human herpesviruses (HVs) (cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], herpes simplex virus [HSV], and human herpesvirus 6 [HHV-6]) in a clinical evaluation of human immunodeficiency virus (HIV)-infected patients with neurological disorders. This method, which has the advantages of being rapid and economical, would be of particular interest for the diagnosis of neurological syndromes caused by more than one HV. We studied 251 CSF samples from 219 patients. HV DNA was demonstrated in 93 (37%) of the CSF samples (34% of the patients). CMV was the HV most frequently detected in our patients (25%), while EBV, VZV, HSV, and HHV-6 DNAs were present in significantly fewer cases (7, 4, 3, and 1%, respectively). When results were compared with the final etiological diagnoses of the patients, the multiplex HV PCR showed high specificity for the diagnosis of CMV and VZV neurological diseases and for cerebral lymphoma (0.95, 0.97, and 0.99, respectively). The sensitivity of the assay was high for CMV disease (0.87), was low for cerebral lymphoma (0.33), and was not evaluable for VZV disease due to the small number of patients with this diagnosis. Nevertheless, detection of VZV DNA had possible diagnostic value in four of the nine cases, and EBV DNA amplification always predicted the diagnosis of cerebral lymphoma in patients with cerebral masses. Detection of HSV DNA was frequently associated with CMV amplification and fatal encephalitis. HHV-6 was not considered to have a pathogenetic role in the three cases in which it was detected. This multiplex HV PCR assay is a specific and clinically useful method for the evaluation of HIV-infected patients with neurological disorders related to HV.

AIDS-Related Opportunistic Infections↗