Multiplex Polymerase Chain Reaction System Research Articles

Development of a 9-locus X-STR multiplex PCR system for genetic analysis of three ethnic populations in China.

X-chromosome short tandem repeats (X-STR) analysis has been confirmed to be effective for kinship testing such as in deficiency paternity cases. The aim of this study was to develop a new multiplex polymerase chain reaction (PCR) system that can simultaneously amplify 9 X-STR loci (GATA172D05, DXS10159, DXS6797, HPRTB, DXS10079, DXS6789, DXS9895, DXS10146 and GATA31E08) in the same PCR reaction, and to obtain the database of the 9 X-STR loci in three ethnic populations in China. The genetic data of 815 (404 females and 411 males) unrelated Han Chinese from Hubei province, and Yi and Zhuang Chinese from Yunnan province were analyzed by using this multiplex system. The results showed that a total of 93 alleles for all these loci were found, and 7 to 20 alleles for each locus were observed. All of the analyzed loci were in agreement with Hardy-Weinberg equilibrium after Bonferroni correction in the three studied populations. The polymorphism information content (PIC) and power of discrimination (PD) in females were 0.6566-0.8531 and 0.8639-0.9684, respectively. Pairwise comparisons of allele frequency distribution showed significant differences in the most of these loci between different populations. The results indicate that this multiplex system is very useful for forensic analysis of different ethnic populations in China.

Multiplex fluorescence quantitative polymerase chain reaction for simultaneous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus.

To construct a multiplex fluorescence quantitative polymerase chain reaction (MFQ-PCR) system to simultaneously detect hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) cDNA, and human immunodeficiency virus (HIV) cDNA to reduce the false positive and false negative results, therefore increasing the efficacy and accuracy of donor blood testing. The feasibility of the MFQ-PCR system was assessed by testing 58 clinical serum samples from hospitalized patients. TaqMan probes designed specifically for the detection of HBV, HCV, and HIV viruses separately were put into the same MFQ-PCR tube and co-amplified with the HBV target DNA, HCV target cDNA, and HIV target cDNA. In this new MFQ-PCR system, the detection limits were about 10 copies for each of standard plasmid DNAs of HBV, HCV, or HIV. In the test of 58 clinical serum samples, of which 23, 16, and 3 were positive for HBV, HCV, and HIV, respectively, by ELISA method, and 16 were normal controls, this new MFQ-PCR system detected 41 of 42 positive samples, and the 16 normal controls were all negative. The rate of consistency between the new system and ELISA was 98.3%. MFQ-PCR has the advantage of simultaneous detection of HBV, HCV and HIV viruses in clinical serum samples with high efficiency and accuracy.

Prevalence of Listeria monocytogenes in ready-to-eat seafood marketed in Thessaloniki (Northern Greece)

Aim: In the current study, a contribution to the knowledge on the prevalence and level of contamination of Listeria monocytogenes in ready-to-eat (RTE) seafood marketed in Thessaloniki (Northern Greece) was provided; the serovar identity of the L. monocytogenes isolates was also determined. Materials and Methods: A total of 132 RTE seafood samples consisting of 74 smoked fish products, 18 salted fish products, 16 dried fish products, 9 raw marinated fish products, 10 cooked marinated cephalopods and 5 surimi crab stick products were analyzed. L. monocytogenes were isolated and enumerated based on ISO 11290-1/A1 and ISO 11290-2/A1 protocols, respectively, and identified using a multiplex polymerase chain reaction (PCR) system utilizing genus and species specific primers. For the identification of serotypes a second multiplex PCR assay was used which clusters L. monocytogenes strains into four major serogroups. Results: Of the samples examined, 11 (8.3%) proved positive for Listeria spp. with 8 (6.1%) yielding L. monocytogenes. Only in one sample of smoked mackerel the level of L. monocytogenes exceeded the legal safety limit of 100 cfu/g set out in Commission Regulation (EC) No. 1441/2007. Serotyping showed higher percentages of isolates belonging to PCR serogroup 3:1/2b, 3b, 7 (46.7%) and serogroup 1:1/2a, 3a (40%) followed by serogroup 4:4b, 4d, 4e (13.3%). Conclusion: This study demonstrated that L. monocytogenes can be isolated from processed RTE seafood products at retail in Thessaloniki (Northern Greece) in low concentrations. However, the presence of this human pathogen in RTE seafood should not be overlooked, but it should be considered as having significance public health implications, particularly among the persons who are at greater risk. Therefore, RTE seafood should be produced under appropriate hygienic and technological conditions since the product does not undergo any treatment before consumption.

Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule

The three most well-known genetically modified (GM) rice lines in China are TT51-1, KMD1, and KF6. The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction (meqPCR) system for simultaneous detection of the three transgenic rice events and to construct a plasmid as the reference molecule for quantitative analysis. Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA. The developed meqPCR was anticipated to detect distinct amplicons as 454, 398, 301, and 250bp from KF6, KMD1, TT51-1, and the rice endogenous reference gene, respectively. The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs, and the sensitivity threshold of the meqPCR was at least 50ng of 0.1% rice DNA for each event when the three transgenic rice events present and with other GM materials together. The constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR. The results indicated that the constructed plasmid was acceptable and suitable for GM rice quantitative analysis.

Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum.

BackgroundAlthough sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed.MethodsThe Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested.ResultsBecause they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively.ConclusionsThe methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries.

Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses

Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10fg/μl of viral RNA under monoplex conditions and 10pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53°C to 63°C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed.

Two quantitative multiplex real-time PCR systems for the efficient GMO screening of food products

According to the EU and Swiss food legislation, only deregulated traits of transgenic plants are allowed to be imported and sold to the consumer. In order to control imports of soya and maize products from retailers, efficient and reliable methods for the detection and quantification are a prerequisite. The screening for specific DNA elements characteristic of transgenic plants is crucial for further analysis and has a major impact on the efficiency of the whole analysis workflow. To allow laboratories to efficiently and reliably screen food products for transgenic plant products, two novel multiplex real-time polymerase chain reaction (PCR) systems were developed and validated. One system determines DNA contents from maize, soya, cauliflower mosaic virus (CaMV) 35S promoter (P35S), NOS terminator from Agrobacterium tumefaciens and CaMV, and the second PCR system simultaneously detects DNA sequences from figwort mosaic virus promoter (PFMV), from bar gene of Streptomyces hygroscopicus, from gene coding for phosphinothricin acetyltransferase (PAT) and from a DNA construct of enolpyruvyl shikimate phosphate synthase gene (CP4-EPSPS) and Arabidopsis thaliana (CPT2). The tests exhibit good specificity and a limit of detection of at least 0.1 % for all analytes.

Differential diagnosis of Goatpox virus in Taiwan by multiplex polymerase chain reaction assay and high-resolution melt analysis

The A32L gene from a Goatpox virus (GTPV) strain isolated from a goat in Yunlin County (Taiwan) displays several substitutions compared with the sequence of the Kenyan GTPV vaccine strain SGP0240 and the Pellor GTPV strain. Samples from the skin lesions on 6 goats with GTPV infection or from goats with Orf virus (ORFV) infection were tested in a multiplex polymerase chain reaction (PCR) system that used primers GPF, GPR1, and GPR2 as well as previously published primers specific for ORFV. These primers were able to amplify either GTPV or ORFV without cross-reactivity. A high-resolution melt analysis (HRMA) was carried out on amplified DNA from the skin lesions of 6 goats with GTPV infection and with the GTPV SGP0240 strain. The results indicated that the melting temperature profiles amplified from samples with Yunlin GTPV infection can be differentiated from the GTPV SGP0240 strain. The findings showed that a successful differential assay for these GTPVs had been developed. Accordingly, both methods can be used to detect and differentiate GTPV isolated from animals that may have either been vaccinated or been infected with a wild strain. The multiplex PCR and HRMA could be used on skin samples of suspected cases to serve as the front-line and confirmative assays, respectively, which will be beneficial to the eradication of GTPV.

Listeria monocytogenes in mussels (Mytilus galloprovincialis) harvested from North Aegean coastal area

One hundred and two samples of mussels (Mytilus galloprovincialis), harvested from approved shellfish coastal water in northern Greece, were screened for the presence and antimicrobial resistance of Listeria monocytogenes. Listeria spp. were isolated according to International Organization for Standardization method 11290-1: 1996/FDAM 1: 2004(E) and identified using a multiplex polymerase chain reaction (PCR) system. The serovar identity of L. monocytogenes isolates was also determined with a multiplex PCR assay. The antimicrobial profile of the isolates was determined by the disk diffusion method. Listeria spp. were present in 8 of 102 samples tested (8%) and only 1 (1%) yielded L. monocytogenes. The isolate identified as L. monocytogenes was defined as serogroup I and found to be resistant to nalidixic acid and streptomycin. In conclusion, this study demonstrated that L. monocytogenes is not commonly found in mussels harvested in the North Aegean Sea, whereas there is a higher possibility of mussels’ contamination with other Listeria species.

Evaluation of 2 Real-Time PCR Assays for In Vitro Diagnostic Use in the Rapid and Multiplex Detection of EGFR Gene Mutations in NSCLC

Activating mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer predict for a favorable clinical response to tyrosine kinase inhibitor therapy. Although Sanger sequencing is a conventional method to detect EGFR gene mutations, multiplex real-time allele-specific polymerase chain reaction (PCR) systems are increasingly used in the routine molecular diagnostic setting. We aim to evaluate 2 proprietary real-time PCR assays (cobas and therascreen) against Sanger sequencing in the detection of EGFR gene mutations. The overall concordance rate between cobas and therascreen assays with Sanger sequencing was 89% and 88%, respectively, and increased to 96% and 98%, respectively, if the mutations not covered were excluded. The cobas assay showed a superior coverage of exon 20 mutations, but L861Q was not targeted. The nature of specimen, DNA integrity, and tumor cell content are factors that affect the assay performance. DNA extracted from cell block and clot of pleural fluid gave rise to 1 invalid call and 1 false-negative result by the cobas assay and 1 missed T790M mutation and 1 false-negative result by the therascreen assay. Both assays are around 5 times more expensive compared with Sanger sequencing in terms of reagent cost. We conclude that both assays prove to be a rapid, simple, and validated method in detecting the most common and clinically significant EGFR gene mutations in non-small cell lung cancer. Although less convenient compared with real-time PCR assays, Sanger sequencing is cheaper in terms of reagent cost and allows the detection of rare or novel EGFR gene mutations.

Molecular characterization and population genetics of non-CODIS microsatellites used for forensic applications in Brazilian populations

Several microsatellites PCR (polymerase chain reaction) multiplex systems (i.e. for simultaneous typing) have been reported for forensic analysis. These include autosomal STR multiplex kits widely used which are commercially available. These commercial kits are generally based on the Combined DNA Index System (CODIS) loci, and a huge volume of genetic population data for the CODIS loci from different ethnic groups has been reported [ 1 Chouery E. Coble M.D. Strouss K.M. Saunier J.L. Jalkh N. Medlej-Hashim M. Ayoub F. Mégarbané A. Population genetic data for 17 STR markers from Lebanon. Leg. Med. 2010; 12: 324-326 Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar , 2 Xu L.N. Hu S.P. Feng G.Y. STR polymorphisms of the Henan population and investigation of central plains Han origin of Chaoshanese. Biochem. Genet. 2009; 47: 569-581 Crossref PubMed Scopus (8) Google Scholar , 3 Grattapaglia D. Schmidt A.B. Costa e Silva C. Stringher C. Fernandes A.P. Pereira M.E. Brazilian population database for the 13 STR loci of the AmpfISTR® Profile Plus™ and Cofiler™ multiplex kits. Forensic Sci. Int. 2001; 118: 91-94 Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar , 4 Egyed B. Füredi S. Angyal M. Balogh I. Kalmar L. Padar Z. Analysis of the population heterogeneity in Hungary using fifteen forensically informative STR markers. Forensic Sci. Int. 2006; 158: 244-249 Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar ]. Nevertheless, there are hundreds of other highly polymorphic STR loci unlinked to the current CODIS loci (non-CODIS markers), which are also useful for forensic genetics [ [5] Asamura H. Ota M. Fukushima H. Population data on 10 non-CODIS STR loci in Japanese population using a newly developed multiplex PCR system. J. Forensic Leg. Med. 2008; 15: 519-523 Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar ]. Analysis of further non-CODIS STR loci may complement information from CODIS loci, offering powerful tools for difficult kinship testing, such as sib-ship testing or testing for deficient paternity cases [ [6] Asamura H. Fujimori S. Ota M. Fukushima H. MiniSTR multiplex systems based on non-CODIS loci for analysis of degraded DNA samples. Forensic Sci. Int. 2007; 173: 7-15 Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar ].

Rapid detection and differentiation of mycobacterial species using a multiplex PCR system

The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection. The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) RESULTS: Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician. Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

Rapid detection of Mycobacterium tuberculosis in clinical samples by multiplex polymerase chain reaction (mPCR)

Although the multi-copy and specific element IS6110 provides a good target for the detection of Mycobacterium tuberculosis complex by PCR techniques, the emergence of IS6110-negative strains suggested that false negative may occur if IS6110 alone is used as the target for detection. In this report, a multiplex polymerase chain reaction (mPCR) system was developed using primers derived from the insertion sequence IS6110 and an IS-like elements designated as B9 (GenBank accession no. U78639.1) to overcome the problem of detecting negative or low copy IS6110 containing strains of M. tuberculosis. The mPCR was evaluated using 346 clinical samples which included 283 sputum, 19 bronchial wash, 18 pleural fluid, 9 urine, 7 CSF, 6 pus, and 4 gastric lavage samples. Our results showed that the sensitivity (93.1%) and specificity (89.6%) of the mPCR system exceeds that of the conventional method of microscopy and culture. The mPCR assay provides an efficient strategy to detect and identify M. tuberculosis from clinical samples and enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.

Use of a multiplex polymerase chain reaction system for enhanced bloodstream pathogen detection in thoracic transplantation

Bloodstream infections (BSIs) constitute a frequent post-transplant complication in thoracic allograft recipients, especially during the early post-surgical period when patients are under intense immunosuppression. Thus, early and accurate identification of the responsible pathogens is of critical importance for patient survival. In this study we investigated the potential clinical utility of a multiplex real-time polymerase chain reaction (PCR) technology (SeptiFast; Roche Diagnostics) for the detection of BSIs in a cohort of thoracic allograft recipients. Our observational study included analysis of 130 blood samples from 30 thoracic allograft recipients (23 heart and 7 lung) using SeptiFast in parallel with blood culture. Samples were drawn when there were clinical and laboratory signs of BSI. The applied molecular assay has been designed to allow direct detection of a wide panel of Gram-positive and Gram-negative bacteria and fungi in blood samples. Real-time PCR yielded concurrent negative and positive results with blood culture methodology in 113 (86.9%) and 5 (3.9%) samples, respectively, with 100% concordance in species identification. SeptiFast identified microorganisms in 9 (6.9%) additional samples that were negative by blood culture. The combined use of SeptiFast and blood culture during the early post-transplant period (<2 months) significantly increased the number of positive samples detected to 17.9% (14 of 78) from 7.7% (6 of 78) detected by blood culture alone (p < 0.05). SeptiFast results were available, on average, within 6 hours from sample collection. The PCR-based SeptiFast test is a valuable addition to the traditional blood culture method for rapid etiologic diagnosis of BSIs in thoracic transplant recipients, especially during the early post-transplant period.

Allelic frequency distributions of 21 non-combined DNA index system STR loci in a Russian ethnic minority group from Inner Mongolia, China

We studied the allelic frequency distributions and statistical forensic parameters of 21 new short tandem repeat (STR) loci and the amelogenin locus, which are not included in the combined DNA index system (CODIS), in a Russian ethnic minority group from the Inner Mongolia Autonomous Region, China. A total of 114 bloodstain samples from unrelated individuals were extracted and co-amplified with four fluorescence-labeled primers in a multiplex polymerase chain reaction (PCR) system. Using capillary electrophoresis, the PCR products of the 21 STR loci were separated and genotyped. A total of 161 alleles were observed in the Russian ethnic minority group, and corresponding allelic frequencies ranged from 0.0044 to 0.5965. The 21 non-CODIS STR loci of the Russian ethnic minority group were characterized by high genetic diversity and therefore may be useful for elucidating the population's genetic background, for individual identification, and for paternity testing in forensic practice.

A New Multiplex Assay of 17 Autosomal STRs and Amelogenin for Forensic Application

This paper describes a newly devised autosomal short tandem repeat (STR) multiplex polymerase chain reaction (PCR) systems for 17 autosomal loci (D1S1656, D2S441, D3S1358, D3S3045, D6S477, D7S3048, D8S1132, D10S1435, D10S1248, D11S2368, D13S325, D14S608, D15S659, D17S1290, D18S535, D19S253 and D22-GATA198B05) and Amelogenin. Primers for the loci were designed and optimized so that all of the amplicons were distributed from 50 base pairs (bp) to less than 500 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. Strategies were developed to overcome challenges that encountered in creating the final assay. The limits of the multiplex were tested, resulting in the successful amplification of genomic DNA range from 0.25–4 ng with 30 PCR cycles. A total of 681 individuals from the Chinese Han population were studied and forensic genetic data were present. No significant deviations from Hardy–Weinberg equilibrium were observed. A total of 180 alleles were detected for the 17 autosomal STRs. The cumulative mean exclusion chance in duos (CMECD) was 0.999967, and cumulative mean exclusion chance in trios (CMECT) was 0.99999995. We conclude that the present 17plex autosomal STRs assay provides an additional powerful tool for forensic applications.

Modified Midi‐ and Mini‐Multiplex PCR Systems for Mitochondrial DNA Control Region Sequence Analysis in Degraded Samples

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185bp. Most mtDNA control region sequences of several 60-year-old and 400-500-year-old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis.

Natural IS711 insertion causing omp31 gene inactivation in Brucella ovis

The present report describes an atypical Brucella ovis strain (Bo10) isolated from the epididymis and testis of an infected ram. Macroscopic and microscopic lesions characteristic for the infection, including positive Brucella immunostaining, were observed within lesions in the genital organs. Compared to other isolates, strain Bo10 required an additional day (a total of 96 hr) of incubation to form visible colonies, showed a distinct carbon source utilization profile, agglutinated only weakly with rough (R) serum, but showed a high capacity for autoagglutination. Isolate Bo10 failed to produce the 1,071-bp fragment in the outer membrane protein (omp) 31 gene-based part of the "Bruce-ladder" multiplex polymerase chain reaction system but did produce a 1,915-bp amplicon, thus presenting a profile similar to Brucella abortus. Sequence analysis of the 1,915-bp fragment revealed an 842-bp long insertion sequence (IS)711 transposon element inserted into the promoter region of the omp31 gene, immediately upstream from the ribosome binding site (-10 box/Pribnow box). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a whole-cell lysate showed the absence in Bo10 of the approximately 31-kDa protein fragment associated with omp31. The results demonstrate a natural inactivation of omp31 and, consequently, the absence of the Omp31 protein in this B. ovis isolate. The novel location of IS711 within the genome of a naturally occurring B. ovis strain supports the hypothesis that IS711 could be an active transposon in this Brucella species.

Validation of the EntericBio Panel II® multiplex polymerase chain reaction system for detection of Campylobacter spp., Salmonella spp., Shigella spp., and verotoxigenic E. coli for use in a clinical diagnostic setting

A total of 717 faeces samples were tested prospectively using the EntericBio Panel II® detection system (Serosep, Limerick, Ireland), in parallel with routine laboratory testing, which combines the EntericBio® system with retrospective culture of each specimen where a target is detected. Discrepancy analysis was conducted using molecular methods. The EntericBio Panel II® assay produced 585 negative and 132 positive results, namely, Campylobacter spp. (n = 66); SLT 1 and/or SLT 2 (n = 64); Salmonella spp. (n = 5); and Shigella spp. (n = 0). Three samples were positive for more than 1 target. Of these results, 4 Campylobacter spp. detections and 4 SLT 1/ SLT 2 detections remained unconfirmed, and the system failed to detect 2 Campylobacter spp. targets detected by routine laboratory detection. The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency were calculated to be 98.4%, 98.7%, 93.9%, 99.7%, and 98.6%, respectively.

Qualitative and Event-Specific Real-Time PCR Detection Methods for Bt Brinjal Event EE-1

Bt brinjal event EE-1 with cry1Ac gene, expressing insecticidal protein against fruit and shoot borer, is the first genetically modified food crop in the pipeline for commercialization in India. Qualitative polymerase chain reaction (PCR) along with event-specific conventional as well as real-time PCR methods to characterize the event EE-1 is reported. A multiplex (pentaplex) PCR system simultaneously amplifying cry1Ac transgene, Cauliflower Mosaic Virus (CaMV) 35S promoter, nopaline synthase (nos) terminator, aminoglycoside adenyltransferase (aadA) marker gene, and a taxon-specific beta-fructosidase gene in event EE-1 has been developed. Furthermore, construct-specific PCR, targeting the approximate 1.8 kb region of inserted gene construct comprising the region of CaMV 35S promoter and cry1Ac gene has also been developed. The LOD of developed EE-1 specific conventional PCR assay is 0.01%. The method performance of the reported real-time PCR assay was consistent with the acceptance criteria of Codex Alimentarius Commission ALINORM 10/33/23, with the LOD and LOQ values of 0.05%. The developed detection methods would not only facilitate effective regulatory compliance for identification of genetic traits, risk assessment, management, and postrelease monitoring, but also address consumer concerns and resolution of legal disputes.