Uniplex PCR Research Articles

New multiplex PCR methods for rapid screening of genetically modified organisms in foods

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

Detection of Pseudomonas aeruginosa by a triplex polymerase chain reaction assay based on lasI/R and gyrB genes.

Pseudomonas aeruginosa is a nosocomial pathogen, which, due to its inherent and acquired resistance to a wide range of antibiotics, causes high mortality rates. Therefore, rapid detection of the bacterium with high specificity and sensitivity plays a critical role in the control of the pathogenic bacterium. The aim of this study was to evaluate the accuracy and specificity of a prompt detection of the bacterium based on a triplex polymerase chain reaction that amplifies the lasI, lasR and gyrB genes. For this purpose, 30 clinical isolates of P. aeruginosa and 30 wound biopsy samples were retrieved from clinical diagnostic laboratories. After the extraction of the chromosomal DNA, the desired genes were amplified using uniplex and triplex PCR with appropriate primers. The specificity of the primers was evaluated by a comparison of the PCR results for P. aeruginosa clinical samples and non-Pseudomonas species control samples. The sensitivity of the primers was determined using a serial dilution of the genomic DNA template (100 ng to 100 fg) and by a comparison of the PCR and bacterial culture results. The results showed that the triplex PCR assay was positive for all of the samples (100%), while the PCR identifications were negative for non-Pseudomonas species. Additionally, at 10(-4) and 10(-5) diluted genomic DNA from P. aeruginosa (10 pg and 1 pg), the triplex PCR test was positive for the Las and gyrB genes in all of the samples, respectively. Based on these results, the designed primers can be used for the rapid, specific and sensitive diagnosis of P. aeruginosa in a triplex PCR assay.

Molecular Characterization of Extended-Spectrum β-Lactamases-Producing Lebsiellapneumoniae Isolated from Clinical Specimens at a Tertiary-Referral Hospital in Denpasar, Bali, Indonesia

Almost no study has been performed in Bali to characterize genotype of ESBL-producing Klebsiellapneumo- niae. The aim of this study was to determine genotype of ESBL produced by K.pneumoniae isolated from clinical specimens in Sanglah General Hospital, Bali, Indonesia. Ninety-seven non-duplicative ESBL-producing K.pneumonia isolates were examined for 16s rRNA, and then with Duplex PCR to detect blaSHV and blaCTX?M genes, and with Uniplex PCR for blaTEM gene. Half of the isolates (50.5%) had coexistences of blaTEM, blaSHV and blaCTX?M genes. Presence of two genes, blaTEM and blaSHV; blaTEM and blaCTX?M; and blaSHV and blaCTX?M???? were 12.4%; 15.5%; and 8.2%, respectively. This study showed the widespread of TEM-, SHV-, and CTX-M- type ESBLs in Bali. Prudent use of antibiotics can save our planet from multidrug resistance microorganisms (MDROs) threat. Keywords: Extended Spectrum Beta-Lactamase, Klebsiellapneumoniae, PCR, Sanglah General Hospital Denpasar.

Two-dye based arrayed primer extension for simultaneous multigene detection in lipid metabolism

BackgroundCardiovascular disease (CVD) is one of the major causes of death worldwide. Numerous genetic risk factors in lipid metabolism, including mutations of LDLR, APOB, and PCSK9, as well as polymorphisms of CETP and APOE, have been found to associate with CVD. MethodsIn this study, a two-dye based arrayed primer extension (APEX) microarray assay for simultaneous multigene (LDLR, APOB, PCSK9, CETP, and APOE) detection was developed. The DNA templates, originating from 1 DNA sample of known genotype and 7 blind DNA samples, were amplified by uniplex PCR. ResultsOptimized conditions for the APEX reaction were determined to include a hybridization temperature of 55°C and a DNA template size of 50–150bp. The total assay including PCR, purification, fragmentation, APEX reaction, and image analysis could be performed in 6h. In total, 48 genotypes were identified among 8 individual DNA samples by APEX analysis. ConclusionsThe data suggest that this APEX microarray offers a robust, fast, and versatile option for screening these genotypes in hypercholesterolemia patients.

Analytical Performance of Multiplex Real-Time PCR for Six Sexually Transmitted Pathogens.

Most organisms that cause sexual transmitted diseases (STDs) are fastidious pathogens that are difficult to detect with conventional microbiological methods and the proportions of multiple infections were noted up to 39.3% among the STI-positive subjects. However, only a few multiplex PCR and multiplex real-time PCR tests that can screen more than six microorganisms that cause STDs have been assessed. A total of 114 endocervical swabs (ThinPrep PAPTEST PreservCyt Solution, Hologic Inc., Marlborough, MA, USA) were collected from healthy Korean women. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), Mycoplasma hominis (MH), Ureaplasma urealyticum (UU), and Trichomonas vaginalis (TV) were detected by uniplex PCR with Seeplex kits and by multiplex real-time PCR with Real-Q Kits (Biosewoom Inc., Seoul, Korea). To evaluate analytical sensitivity, plasmids containing target genes from CT, NG, MG, MH, UU, and TV were serially diluted five times with saline buffer and replicated eight times per dilution. Real-Q STIs Kit assays showed 100% sensitivity for detecting MH, MG, CT, TV, NG and 94.1% sensitivity for detecting UU. In addition, it showed 100% specificity for UU, MH, MG, CT, TV, and NG. The analytic sensitivity of UU (95% probit = 17.3 copy/μL, 95% CI = 11.6 to 138.6) and MH (95% probit = 30.9 copy/μL, 95% CI = 20.6 to 169.9) was relatively lower than for others pathogens. Thus, the cutoff Ct value of < 45 for UU and MH and a cutoff Ct value of < 38 for CT, MH, NG, TV could minimize differences in detection limit among the six STIs (95% probit values = 5.3 to 14.6) and to optimize overall diagnostic performance. For medical applications of a multiplex real-time PCR assay, one kind of cutoff value, which is according to manufacturer's instructions, was generally used without the consideration of lowest actual detectable concentration of each target substance. However, analytical performance at the low concentration limit often defines the ability of the test to diagnose disease and determine treatment endpoints. Therefore, suitable cutoffs for negative or positive screens by multiplex real-time PCR should be evaluated for accurate diagnosis.

Bead-based suspension array for simultaneous differential detection of five major swine viruses.

A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2-22 copies/μL (corresponding to 0.5-6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.

Duplex Real Time PCR for Simultaneous Detection of Salmonella Species and Listeria Monocytogenes in Frozen Meat

In this study, duplex real-time PCR approach with melting curve analysis is presented for the detection of Listeria monocytogenes, which are important food-borne bacterial pathogens usually present in imported frozen meat. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic Tm values demonstrating the specific and efficient amplification of the two pathogens;73.2°C and 81.54°C for L. monocytogenes and Salmonella spp., respectively, the duplex, real-time PCR was equally sensitive to uniplex real-time PCR, using same amounts of purified DNA. When our established duplex real-time PCR assay was applied to artificially inoculated imported frozen meat the detection limit was 103 CFU/g for each of these pathogens without enrichment. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquot. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.

Lovebirds and Cockatiels Risk Reservoir of Cryptococcus neoformans, a Potential Hazard to Human Health

Lovebirds and Cockatiels Risk Reservoir of Cryptococcus neoformans, a Potential Hazard to Human Health Lovebirds and cockatiels are potential carriers and/or transmitters of zoonotic diseases. Some of them could have an important impact for human health, In Egypt, the role of these birds in dispersing C. neoformans is not well documented, which evoked a high need to investigate the environmental ecology of this fungus in order to establish surveillance programs and applying the preventive measures for this pathogen infection. The C. neoformans prevalence and role of pet birds in spreading this fungal pathogen in Egypt was illustrated in this study. Two hundred Cockatiels and lovebirds excreta were collected from captive birds. The recovered isolates of C. neoformans species were identified by molecular identification using capsular gene specific primer CAP64. The subtyping of isolates was performed by multiplex PCR using CNa-70S/A -CNa-49S/A. Four isolates (3 and 1) from lovebirds & Cockatiels respectively were subjected to sequence analysis of Internal Transcribed Spacer (ITS) regions. Alternatively the fungal isolates were analyzed by PCR fingerprinting with uniplex PCR amplification using an oligonucleotide (GTC)5. From this study it was concluded that, the excreta of these birds can play a role as a risk reservoir of C. neoformans in domestic and public environments and enhance their zoonotic importance to human.

Non-invasive genetic diagnosis of Duchenne Muscular Dystrophy probands using salivary DNA

Duchenne Muscular Dystrophy (DMD) is the most common fatal X-linked recessive disorder, which means that the disease is carried by mother but usually appears in boys. Since muscle wasting occurs in these children, it is difficult to draw blood from them for the DNA test and also it is an invasive technique. The present study is to bring out the non-invasive method of DNA test for the children. Saliva also harbors a wide spectrum of genetic data that can be used for genetic research and clinical diagnostic applications. Saliva samples from several DMD patients and non DMD patients (as control) were collected and the total DNA was extracted by the modified method and their quality were also determined by spectroscopic method. The resultant DNA was of better quality and good for amplification of the DMD exons by PCR, both uniplex and multiplex. But only bands of less intensity were seen on the gel which may be due to PCR inhibitors and this was further confirmed by PCR method. DNA isolated by this method does not have PCR inhibitors and this was shown by the fact that both multiplex and uniplex PCR’s showed a significant level of amplicons. This study has shown that saliva could be used as a source of genomic DNA for use in PCR based genetic analysis. If standardized, this could be cost effective and less invasive and will benefit the children and old patient too for the diagnosis of all genetic disorders. Thus, this study may be used for the analysis of disease diagnosis and genetic disorder using saliva DNA.

Detecção de genes toxigênicos, susceptibilidade antimicrobiana e antagonismo in vitro de Staphylococcus spp. isolados de queijos artesanais

Staphylococcus spp. isolated from samples of Minas cheese traditionally manufactured following artisan procedures were identified using molecular techniques and further analyzed using PCR and specific primers for the detection of classic enterotoxins (SEA, SEB, SEC, SED, and SEE) and toxic shock syndrome toxin-1 (TSST-1). Specific sea, sec, sed, and see genes were identified using multiplex PCR, whereas seb and tst genes were detected by uniplex PCR. In vitro antagonism with Lactobacillus spp. was evaluated to assess antimicrobial susceptibility. Classic enterotoxins and TSST-1 genes were not detected. The antimicrobials sulfonamide, penicillin, ceftzadime, and oxacillin showed higher resistance rates in the antibiogram (100%, 80%, 60%, and 40%, respectively), whereas other antimicrobials were effective in percentages above 70%. Lactobacillus spp. were able to inhibit Staphylococcus spp. in vitro. Thus, our results indicated that the isolated Staphylococcus spp. were sensitive to the most common antimicrobials tested and were inhibited by Lactobacillus spp.

Chlorpyrifos-induced changes in the antioxidants and fatty acid compositions of Chroococcus turgidus NTMS12.

This study investigated the effect of pesticide chlorpyrifos (CP) on a freshwater cyanobacterium Chroococcus turgidus NTMS12. The changes in chlorophyll-a, proline, superoxide dismutase (SOD), catalase (CAT) activities and fatty acid composition of the test organism were analysed. Organism was grown at 6, 9 and 12 mg l(-1) of CP, and based on the chlorophyll-a content, 6 mg l(-1) of CP was found to be the tolerable concentration. Hence, 6 mg l(-1) of CP was taken to evaluate the concentration of proline and activities of SOD and CAT at 48-h exposure. The changes in the fatty acid profile were analysed after 7 days of exposure. Upon pesticide exposure, increased concentration of proline and activities of SOD and CAT were found. Significant changes in fatty acid profile have also been observed. However, polyunsaturated fatty acid content was decreased in treated cultures when compared with the untreated control. Changes in biochemical activities indicate that cyanobacteria C. turgidus NTMS12 undergo adaptive changes against CP-induced oxidative stress. : Chlorpyrifos induces oxidative stress in Chroococcus turgidus NTMS12. A strong inference was made on increased activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and proline content and lowering the level of unsaturated fatty acids under the pesticide-exposed condition. These significant changes are the defence mechanisms against the oxidative stress. Thus, this organism holds great promise in resisting toxic pesticide.

Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria.

Species of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (mPCR) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus-specific primers instead of species-specific ones, the current mPCR covered much more target bacterial species compared with previously reported species-specific mPCR methods. The specificity of the four putative genus-specific primers was validated experimentally while used exclusively (uniplex PCR) or combined (mPCR) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: Aeromonas (875bp), Vibrio (524bp), Edwardsiella (302bp) and Streptococcus (197bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The mPCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The mPCR detection limits for each target bacterial genera were 50 colony-forming units (CFU) in pure culture and 100CFU in fish tissue samples. In conclusion, the mPCR assay was proven to be a powerful alternative to the conventional culture-based method, given its rapid, specific, sensitive and reliable detection of target pathogens. The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus-specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The mPCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species-specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This mPCR assay provides a rapid, specific and sensitive tool for the detection or identification of common fish pathogenic bacteria in aquaculture practice.

Lower prevalence of hlyD, papC and cnf-1 genes in ciprofloxacin-resistant uropathogenic Escherichia coli than their susceptible counterparts isolated from southern India.

The study was conducted to determine the association of the hlyD, papC and cnf-1 virulence genes with drug resistance in uropathogenic Escherichia coli (UPEC) isolated from cases of urinary tract infection (UTI). A total of 193 E. coli strains isolated from symptomatic cases of UTI in a tertiary care teaching hospital in Raichur, Northern Karnataka, India were included in the study. The antibiotic susceptibility pattern was determined by Kirby-Bauer's Disk Diffusion method, and the strains resistant to any of the third generation cephalosporins tested were further confirmed for extended-spectrum beta-lactamase (ESBL)-production by an E-strip test. Genotypic virulence markers, namely, hlyD, papC and cnf-1, were detected by the uniplex PCR method and the phylogenetic characterization was performed by a multiplex PCR assay. The majority of the E. coli isolates belonged to the B2 phylogenetic group were significantly associated with ciprofloxacin-sensitivity and non-ESBL production (p<0.05). An increased prevalence of ciprofloxacin-sensitive strains over ciprofloxacin-resistant strains were observed among the UPEC isolates harboring the papC (72.9% vs. 40.2%; p<0.001), hlyD (43.7% vs. 21.6%; p<0.001) and cnf-1 (30.2% vs. 12.3%; p<0.05) genes. The presence of a multivirulent gene in the non-ESBL E. coli strains (44.5%) was significantly higher (p<0.05) than in the ESBL-producing strains (21%). Among the UPEC isolates, the predominant B2 phylogenetic group was significantly associated with the ciprofloxacin-sensitive strains, as well as with the non-ESBL E. coli strains. The genotypic virulence markers of UPEC were associated with ciprofloxacin-sensitivity, and a significant number of the non-ESBL strains harbored multivirulent genes. The relationship between the presence of the virulence genes and ESBL production was complex and warrants further intensive studies.

Parallel tagged amplicon sequencing of transcriptome‐based genetic markers for Triturus newts with the Ion Torrent next‐generation sequencing platform

Next-generation sequencing is a fast and cost-effective way to obtain sequence data for nonmodel organisms for many markers and for many individuals. We describe a protocol through which we obtain orthologous markers for the crested newts (Amphibia: Salamandridae: Triturus), suitable for analysis of interspecific hybridization. We use transcriptome data of a single Triturus species and design 96 primer pairs that amplify c. 180 bp fragments positioned in 3-prime untranslated regions. Next, these markers are tested with uniplex PCR for a set of species spanning the taxonomical width of the genus Triturus. The 52 markers that consistently show a single band of expected length at gel electrophoreses for all tested crested newt species are then amplified in five multiplex PCRs (with a plexity of ten or eleven) for 132 individual newts: a set of 84 representing the seven (candidate) species and a set of 48 from a presumed hybrid population. After pooling multiplexes per individual, unique tags are ligated to link amplicons to individuals. Subsequently, individuals are pooled equimolar and sequenced on the Ion Torrent next-generation sequencing platform. A bioinformatics pipeline identifies the alleles and recodes these to a genotypic format. Next, we test the utility of our markers. baps allocates the 84 crested newt individuals representing (candidate) species to their expected (candidate) species, confirming the markers are suitable for species delineation. newhybrids, a hybrid index and hiest confirm the 48 individuals from the presumed hybrid population to be genetically admixed, illustrating the potential of the markers to identify interspecific hybridization. We expect the set of markers we designed to provide a high resolving power for analysis of hybridization in Triturus.

Application of PCR-Based Methods for Rapid Detection of Corn Ingredients in Processed Foods

A rapid and accurate detection of corn (Zea mays L.) ingredients in processed foods is important for food safety and quality assurance. This study aimed to develop PCR-based approach for fast screening of the corn in foodstuffs. To this purpose a new PCR-based DNA marker specific to the corn zein gene was developed, three uniplex PCR methods and one triplex PCR system targeting invertase and zein genes were compared. Different corn-derived foodstuffs such as: flour, chips, flakes and snacks were investigated. Analysis of PCR products by agarose gel electrophoresis demonstrated that multiplex PCR method represents the most reliable and rapid tool for identification of corn ingredients in highly processed foods.

Development and evaluation of a multiplex PCR for eight plasmid-mediated quinolone-resistance determinants

The objective of this study was to develop and validate an expanded multiplex PCR assay for the simultaneous detection of eight plasmid-mediated quinolone-resistance determinants in Enterobacteriaceae. Primers were designed to amplify conserved fragments of qnrABCDS, qepA, oqxAB and aac(6')-Ib-cr genes and were optimized in uniplex and multiplex PCR assays with control template DNA. The assay was used to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in 174 ciprofloxacin-resistant and 43 ciprofloxacin-susceptible extraintestinal pathogenic Escherichia coli isolates. Each resistance gene could be detected alone and in combination. PMQR determinants were detected in 65 ciprofloxacin-resistant isolates (37 %) and one ciprofloxacin-susceptible isolate (2 %). Prevalences of the identified determinants were: aac(6')-Ib-cr, 34.5 %; qnrS, 1.1 %; qepA, 1.1 %; and oqxAB, 0.6 %. In conclusion, we developed an eight-target multiplex PCR for the accurate detection of PMQR genes and confirmed that PMQR prevalence remains low among human Escherichia coli clinical isolates in the UK.

P2.050 Development of Molecular Beacon Based Diagnostic Assay For Detection of Neisseria Gonorrhoeae and Chlamydia Trachomatis

BackgroundDue to absence of cost effective and rapid diagnostic test syndromic management of Neisseria and Chlamydia was recommended in developing countries. Being nonspecific such a strategy has resulted in over...

Uniplex and duplex PCR detection of geminivirus associated with potato apical leaf curl disease in India

Apical leaf curl disease has emerged as a new disease in potato during the last decade in India due to a change in planting date and an increased whitefly population. Its incidence is on the rise threatening the cultivation of potato across the country. Hence, a PCR assay was developed for the detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV-Potato) which is the causal agent of apical leaf curl disease in potato. Primers specific to the coat protein (AV1) and replicase (AC1) gene regions were designed and used for standardization of the PCR. Some of the primers (LCVCPF1/LCVCPR1, LCVREPF2/LCVREPR2, LCrep1F/LCrep2R) could detect the virus in 2.4–0.24pg of total DNA of infected plant. A duplex PCR assay was optimized with the selected coat protein gene specific primers and primers specific to potato urease gene, a housekeeping gene served as an internal check. The suitability of these primers was examined for detection of the virus in 80 potato apical leaf curl disease samples from 11 different potato growing states of India and also from micro-plants grown in tissue culture. The selected coat protein primer pair (LCVCPF1/LCVCPR1) was found to be conserved in all 80 isolates except for a few isolates, which had a single nucleotide substitution in the forward primer sequence. These substitutions did not interfere with amplification of the coat protein gene. The primers could detect the virus using a print-capture PCR assay both in the presence and absence of an internal control. These results indicate the robustness of the PCR assay for virus indexing of mother stocks in the seed production system.

Multiplex PCR (Polymerase Chain Reaction) Assay for Detection of E. coli O157:H7, Salmonella sp., Vibrio cholerae and Vibrio parahaemolyticus in Spiked Shrimps (Penaeus monodon)

The coastal aquaculture mainly shrimps constitute major export sector in Bangladesh and is increasingly shaped by international trade conditions and by national responses to those stringent quality and safety standards. PCR based validated methods for detection of major bacterial pathogens in shrimp might be very useful tool for ensuring quality and safety standards of exportable shrimps. The objective of this study was to evaluate overall performance (sensitivity and specificity) of the multiplex PCR assay for detection of Vibrio cholerae, Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli O157:H7 from spiked shrimp samples. The targeted genes were ompW for V. cholerae, tdh for V. parahaemolyticus, sefA for Salmonella spp. and hlyEHEC for E. coli O157:H7. The genomic DNA was extracted by using standard method and amplified accordingly. Sensitivity of the assay was tested by inoculating the shrimp homogenate with viable cells of laboratory references strains (target pathogens). The genes were amplified individually both from culture homogenate and spiked samples. Twenty different uniplex and multiplex PCR assay were performed; the results showed that the sensitivity and specificity of multiplex PCR are comparable to that of the results of uniplex PCR for the samples. DNA extracted from shrimp samples spiked with non-target pathogen (Bacillus cereus, Shigella flexneri and Staphylococcus aureus) yielded negative results.

Application of three uniplex polymerase chain reaction assays for the detection of atypical bacteria in asthmatic patients in Kuwait

Respiratory infections are known to exacerbate wheezing in many asthmatic patients. We aimed to use molecular methods for the fast detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila in respiratory specimens from asthmatic patients in Kuwait. We used uniplex PCR assays to detect the three atypical bacteria in clinical specimens from 235 asthmatic and non-asthmatic patients in Kuwait. A regression analysis was used to identify the risk factors related to the bacterial type. Group comparisons for similarity were conducted and correlation coefficients were calculated using SPSS statistical software. The detection limits using uniplex PCR for C. pneumoniae, L. pneumophila and M. pneumoniae were approximately 1pg, 2.4fg and 12pg of DNA, respectively. M. pneumoniae PCR positivity was more common in asthmatic patients (15%) than in non-asthmatic subjects (9%) (P<0.05). A marked difference was observed between patients with acute asthma exacerbation (11%) and patients with chronic (stable) asthma (7%) among Kuwaiti patients; these percentages were 16% for non-Kuwaiti acute asthma patients and 14% for non-Kuwaiti chronic asthma patients (P<0.201). There was a weak positive correlation between asthma severity and PCR positivity for M. pneumoniae. The PCR results for C. pneumoniae and L. pneumoniae were found to be statistically insignificant. The results of this study suggest that infection with M. pneumoniae may be related to the exacerbation of asthma symptoms and could possibly be a factor that induces wheezing.