Multiplex Polymerase Chain Reaction Protocol Research Articles

Development of 14 minisatellite markers for the citrus canker bacterium, Xanthomonas citri pv. citri

We screened the genome of Xanthomonas citri pv. citri strain 306 for tandem repeats. A multiplex polymerase chain reaction protocol was used to assess the genetic diversity of 239 strains of X. citri pv. citri from Asia. The total number of alleles per locus ranged from three to 20. Using pooled data sets, 223 different haplotypes were identified. Successful amplifications were obtained at most loci for seven other X. citri pathovars. This typing scheme is expected to be useful at different spatial scales for population studies of pathovars of X. citri, several of which cause plant diseases of economic importance.

Pitfalls in TCR gene clonality testing: teaching cases

Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.

Diseño y evaluación de una reacción de polimerasa en cadena (RPC) múltiple, para la identificación de bacterias causantes de meningitis aguda en líquido cefalorraquídeo de niños chilenos

Acute bacterial meningitis (ABM) is a serious disease that needs rapid diagnosis for an accurate treatment. The most important etiological agents are: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type b. Overall pathogen detection rate in patients with ABM in Chile is 83%.To evaluate a Polymerase Chain Reaction (PCR) protocol for simultaneous detection of several pathogens in patients with ABM.We designed and evaluated a multiplex PCR protocol for simultaneous specific genes identifications of S pneumoniae ( inverted exclamation markytA and ply genes), N meningitidis (ctrA, crgA) and H influenzae (bexA) in cerebrospinal fluid (CSF) samples from pediatric patients with suspected diagnosis of ABM. Sensitivity, specificity and minimum detection levels of DNA were determined. Amplifications ofrDNA 16S gene was done to confirm extraction of bacterial DNA.Ninety nine CSF samples were studied, 90 from children with fever and negative CSF culture, and 9 from ABM and positive culture patients. The PCR protocol had a sensitivity of 89%, specificity of 100%, positive predictive value 100% and negative predictive value 99%.We observed a high concordance (89%) between bacteriological cultures and the PCR protocol results. This diagnostic tool could increase identification of agents in specific settings such as patients previously treated with antibiotics.

Prevalence of Escherichia coli O157:H7 on dairy farms in Shiraz, Iran by immunomagnetic separation and multiplex PCR

Summary To identify the reservoirs of shiga toxin-producing Escherichia coli O157, sensitive detection and isolation methods are necessary. The sensitivity of traditional culture methods can be improved significantly by the inclusion of an immunoconcentration step, resulting in less false-negative results. In this study, enrichment procedure and immunomagnetic separation (IMS) were compared for use in conjunction with a multiplex polymerase chain reaction (M-PCR) method for detection of genes stx1, stx2, eaeA and hlyA. A total number of 975 faecal samples were collected from 26 dairy farms in Shiraz area, Shiraz, southern Iran. The samples were cultured at 37°C for 18–24 hrs in modified tryptic soy broth (m-TSB). Each of five enriched samples were pooled and examined in two ways—direct PCR and IMS. The detection limit of the M-PCR protocol for seeded E. coli O157:H7:ATCC:43895 in m-TSB without stool was 1.23 × 10 2 CFU/ml, whereas it was 1.23 × 10 6 CFU/ml with enriched faecal sample. In direct PCR of enriched samples, no positive sample was detected. However, in IMS of enriched samples one specimen was positive. The prevalence of E. coli O157:H7 in faeces of cows in examined farms was 0.51% and the herd prevalence was 3.86%. Isolation of this serotype from faecal samples indicates that cattle are reservoirs of this pathogen and potentially a source of human infection. This finding is of considerable public health importance.

Immunoglobulin/T-cell receptor clonality diagnostics

Clonality testing in lymphoid malignancies has become technically relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for Ig/T-cell receptor (TCR) gene analysis. Expertise with clonality diagnostics and knowledge about the biology of Ig/TCR recombination are essential for correct interpretation of the Ig/TCR clonality data. Several immunobiologic and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this review. Furthermore, the need to integrate the molecular data with that from (hemato-)pathology, and preferably also flowcytometric immunophenotyping for appropriate interpretation, is discussed. Such an interactive, multidisciplinary diagnostic model guarantees integration of all available data to reach the most reliable diagnosis.

Detection ofEutypa lataandEutypella vitisin Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.

Comparison of Campylobacter jejuni PFGE and Penner subtypes in human infections and in water samples from the Taieri River catchment of New Zealand

To determine the degree of overlap in strain types of Campylobacter jejuni isolated from clinical cases and water samples from the Taieri catchment in the South Island of New Zealand. Thermophilic Campylobacter were collected from human cases of infection, the main stem of the Taieri River and streams within distinct land-use types over a 1-year period. Campylobacter jejuni (187 isolates) and Campylobacter lari (four isolates) were identified using a multiplex polymerase chain reaction protocol. Isolates were typed by the Penner method and pulsed-field gel electrophoresis (PFGE) utilizing two restriction endonucleases. Several serotypes and PFGE types occurred in both water samples and clinical cases when the restriction profiles for each enzyme were considered separately. However, when PFGE profiles and serotyping were combined, there was no overlap between Camp. jejuni types from water and clinical cases. The results of this study indicate that recreational water in the Taieri catchment is not a major source of campylobacteriosis in the Dunedin area. This study suggests the risk of acquiring campylobacteriosis from surface waters in the Taieri catchment is considerably lower than previously predicted and highlights the necessity of using two endonucleases in PFGE typing.

ABO blood group in Kuwaitis: detailed allele frequency distribution and identification of novel alleles

The ABO blood group is clinically the most important blood group system and can now be genotyped easily by DNA-based methods without family studies. Samples (n = 166) from a Kuwaiti population were phenotyped by standard serologic techniques for the ABO blood group and genotyped for the ABO locus by an established multiplex polymerase chain reaction protocol followed by single-strand conformation polymorphism (SSCP) analysis. Nonstandard SSCP patterns were investigated by DNA sequencing of exons 6 and 7 and, if necessary intron 6. Standard SSCP patterns identified six classical alleles in this population: A101 (0.1115), A102 (0.0181), A201 (0.0301), B101 (0.1627), O101 (0.3103), and O201 (0.2500). One A, 1 B, and 8 O variant alleles were identified (total frequency, 0.1175). All variant alleles were each present in one or two chromosomes (< or =0.0060) in our samples except O109 (0.0813). Three of these 10 variant alleles were novel alleles defined by newly identified single-nucleotide polymorphisms in exon 7 (527G>A, 687C>T, and 1116G>A). One new base substitution result in amino acid change. This is the first study reporting the detailed distribution of ABO alleles and genotypes in Kuwaitis. Sixteen alleles were identified, including 3 novel alleles.

Polymorphisms of the GSTM1 and GSTT1 genes in patients with allergic diseases in the Czech population

Allergic diseases belong to the most common chronic disorders affecting mankind and their prevalence in population is increasing. Several studies have indicated that oxidative stress impairs pulmonary function and makes existing asthma worse. Members of the glutathione-S-transferase (GST) superfamily of genes are important in protection of cells from reactive oxygen species. Relationships among allergic diseases including asthma and variations in the GST mu (GSTM1) and GST theta (GSTT1) genes were investigated in 1,006 Caucasian subjects. The multiplex polymerase chain reaction protocol was used for simultaneous amplification of both genes for molecular analysis. Genotype frequencies among patients and controls were assessed and the associations of the genotypes with intermediary phenotypes of allergy were statistically determined. The frequencies of GST null genotypes did not differ significantly between patients with allergic diseases (or asthma alone) and healthy controls. However, when compared with patients homozygous or heterozygous for GSTM1 functional allele, asthmatics carrying both GSTM1 null alleles displayed significantly worse lung function, assessed by forced expiratory volume in 1 s/forced vital capacity (FEV(1)/FVC) ratio (Tiffenau index), (P < 0.01). Genetic polymorphisms of the GSTM1 and GSTT1 genes, both individually and in combination, were not associated with the development of allergic diseases including asthma in the Czech population, the GSTM1 gene variability, however, may influence lung functions in our asthmatics.

Investigation of erythromycin and tetracycline resistance genes in methicillin-resistant staphylococci

In this study, erythromycin [ erm(A) and erm(C)] and tetracycline [ tet(K) and tet(M)] resistance genes were investigated by multiplex polymerase chain reaction (PCR) in a total of 56 methicillin-resistant ( mecA+) staphylococcal hospital isolates, 28 of which were determined to be Staphylococcus aureus (MRSA) and the other 28 were coagulase-negative staphylococci (MRCNS). Internal control primers amplifying a specific fragment of 16S rDNA of staphylococci were included in the multiplex PCR protocol to ensure the efficacy of amplification and to determine any PCR inhibition. No resistance genes were detected in 5 of 56 (8.9%) isolates in the study. In the study, tet(K) genes were detected widely (42.9%) in MRCNS, whilst tet(M) genes were detected in MRSA (50.0%). Regarding the erythromycin resistance genes, whilst erm(A) genes were detected in most (71.4%) MRSA isolates, detection rates of erm(C) genes were the same (64.3%) both in MRCNS and MRSA. The resistance rates for tetracycline and erythromycin were 57.1% and 78.6%, respectively, in MRSA isolates. In conclusion, in this study, the multiplex PCR technique including an internal control is shown to be a fast, sensitive, reliable, practical, reproducible and economic technique for the detection of erythromycin and tetracycline resistance in staphylococcal isolates.

Rapid Detection of Virulence-Associated Genes in Avian Pathogenic Escherichia coli by Multiplex Polymerase Chain Reaction

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.

Additional microsatellites for Sparus aurata and cross‐species amplification within the Sparidae family

AbstractSix new microsatellite loci were isolated and characterized in 32 individuals from a farm population of gilthead seabream (Sparus aurata). Expected heterozygosity at all loci was high, ranging from 0.835 to 0.958 with between 10 and 27 alleles per locus. A multiplex polymerase chain reaction protocol was developed using four of the loci. Cross‐species amplification of the loci was tested in six species of the Sparidae family and four loci were successfully amplified in two or more related species.

Detection of Sorbitol-Negative and Sorbitol-Positive Shiga Toxin-ProducingEscherichia coli,Listeria monocytogenes,Campylobacter jejuni, andSalmonellaspp. in Dairy Farm Environmental Samples

Six visits were conducted to four dairy farms to collect swab, liquid, and solid dairy farm environmental samples (165 to 180/farm; 15 sample types). The objective of the study was to determine on-farm sources of Campylobacter jejuni, Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC), which might serve as reservoirs for transmission of pathogens. Samples were analyzed using mostly U.S. Food and Drug Administration's Bacteriological Analytical Manual protocols; however, Salmonella spp., L. monocytogenes and STEC were co-enriched in universal pre-enrichment broth. Campylobacter jejuni were enriched in Bolton broth containing Bolton broth supplement. Pathogens were isolated on agar media, typed biochemically, and confirmed using multiplex polymerase chain reaction protocols. Campylobacter jejuni, Salmonella spp., L. monocytogenes, Sorbitol-negative (SN)-STEC O157:H7, and sorbitol-positive (SP)-STEC, respectively, were isolated from 5.06%, 3.76%, 6.51%, 0.72%, and 17.3% of samples evaluated. Whereas other pathogens were isolated from all four farms, SN-STEC O157:H7 were isolated from only two farms. Diverse serotypes of SP-STEC including O157:H7, O26:H11, O111, and O103 were isolated. None of the five pathogen groups studied were isolated from bulk tank milk (BTM). Most pathogens (44.2%) were isolated directly from fecal samples. Bovine fecal samples, lagoon water, bedding, bird droppings, and rat intestinal contents constituted areas of major concern on dairy farms. Although in-line milk filters from two farms tested positive for Salmonella or L. monocytogenes, none of the pathogens were detected in the corresponding BTM samples. Good manure management practices, including control of feral animals, are critical in assuring dairy farm hygiene. Identification of on-farm pathogen reservoirs could aid with implementation of farm-specific pathogen reduction programs.

Characterizing avian Escherichia coli isolates with multiplex polymerase chain reaction.

Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos,and the isolate of healthy birds that contained all the genes killed the most embryos amongthis group. These genes were not found among the non-E. coli isolates tested, demonstrating the procedure's specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.

Combining multiplex and touchdown PCR for microsatellite analysis.

An improved nonradioactive polymerase chain reaction (PCR)-based method for simultaneous amplification of multiple loci of microsatellites has been developed as a rapid way to screen for microsatellites (). The approach, termed multiplex-touchdown PCR (MT-PCR), is performed in a single PCR tube by combining touchdown (, , , ) and multiplex () PCR protocols. The touchdown format is used to improve the specificity and the quality of amplification, that is, only DNA bands of an expected size are present as the major PCR bands observed on the nondenaturing polyacrylamide gels, thereby overcoming the presence of differently sized background bands (a ladder-like problem). The multiplex strategy is used so that simultaneous amplification of multiple microsatellite loci is achieved. In this chapter, we describe the MT-PCR strategy that has been successfully used for simultaneous amplification of up to three mouse microsatellites by choosing primer pairs with the corresponding touchdown-PCR parameters. The MT-PCR is very useful for genotyping hybrid mice, provided the allelic size difference between two parental genotypes is amenable to separation by gel electrophoresis. In principle, this MT-PCR should be applicable to similar studies in other species, including humans.

Direct Detection of Insertion/Deletion Polymorphisms in an Autosomal Region by Analyzing High-Density Markers in Individual Spermatozoa

Direct polymerase chain reaction (PCR) detection of insertion/deletion (indel) polymorphisms requires sample homozygosity. For the indel polymorphisms that have the deletion allele with a relatively low frequency in the autosomal regions, direct PCR detection becomes difficult or impossible. The present study is, to our knowledge, the first designed to directly detect indel polymorphisms in a human autosomal region (i.e., the immunoglobulin V(H) region), through use of single haploid sperm cells as subjects. Unique marker sequences (n=32), spaced at approximately 5-kb intervals, were selected near the 3' end of the V(H) region. A two-round multiplex PCR protocol was used to amplify these sequences from single sperm samples from nine unrelated healthy donors. The parental haplotypes of the donors were determined by examining the presence or absence of these markers. Seven clustered markers in 6 of the 18 haplotypes were missing and likely represented a 35-40-kb indel polymorphism. The genotypes of the donors, with respect to this polymorphism, perfectly matched the expectation under Hardy-Weinberg equilibrium. Three V(H) gene segments, of which two are functional, are affected by this polymorphism. According to these results, >10% of individuals in the human population may not have these gene segments in their genome, and approximately 44% may have only one copy of these gene segments. The biological impact of this polymorphism would be very interesting to study. The approach used in the present study could be applied to understand the physical structure and diversity of all other autosomal regions.

Rapid Detection of Salmonella in Water Samples by Multiplex Polymerase Chain Reaction

A rapid protocol for detecting Salmonella species in water samples using the polymerase chain reaction (PCR) technique is described in this paper. Salmonellae, the etiological agents for typhoid fever, salmonellosis, and gastrointestinal infections, require more than 30 hours to be detected in water samples using current standard protocols. In epidemic conditions, where detection time is crucial, the multiplex PCR protocol developed can give results within 5 hours of collection of water samples. This latter protocol uses a gradient temperature program that allows simultaneous amplification of five different loci in a single reaction. The target loci used were invA, phoE, spvA, spvB, and 16s rDNA gene. The selected primers in the reactions allow the detection of a broad range of pathogenic Salmonella, whereas 16s rRNA locus was included as a reaction control for raw water samples devoid of Salmonella. The protocol has been successfully tried on standard strains and on samples collected from river water.

Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples.

Due to the clinical importance of differentiating the two species of the Entamoeba histolytica/Entamoeba dispar complex, we developed a multiplex polymerase chain reaction (PCR) method that overcomes time-consuming and laborious procedures. We report here a DNA extraction protocol using non-fixed stool samples that avoid long lysis-incubation periods through the combined use of zirconium beads and a lysis-supporting buffer. We characterized 49 of 52 stool specimens from Cuban patients with amoebiosis. Among them, 36 (75.5%) were infected only with E. dispar (the nonpathogenic species), while 13 (24.5%) displayed a mixed infection with both E. dispar and E. histolytica. The multiplex PCR protocol showed a specificity of 1.00 and a sensitivity of 0.94. Furthermore, the entire procedure can be performed in one day. This approach is therefore reliable and applicable in the field for epidemiologic studies.

Glutathione S-transferase T1 and M1 gene defects in ovarian carcinoma

Glutathione S-transferases (GSTs) M1 and T1 are known to be polymorphic in humans. Both polymorphisms are due to gene deletions, which are responsible for the existence of null genotypes. The gene defect of GSTT1 has been reported to be associated with an increased risk of myelodysplastic syndromes, astrocytoma and meningioma. A lack of GSTM1 was associated with tobacco smoke-induced lung and bladder cancer. In this study we examined whether the GSTT1 and/or GSTM1 homozygous null genotypes were associated with an increased risk of ovarian cancer using a multiplex polymerase chain reaction protocol. The GSTT1 null genotype was observed in 14% of the control subjects that had never suffered from neoplastic disease ( n=115) and in 16% of the patients affected with ovarian cancer ( n=103, OR 0.87, 95% CI 0.39–1.92, P=0.73). A lack of GSTM1 was observed in 38% of the control subjects and in 46% of the patients (OR 0.77, 95% CI 0.44–1.32). This difference was not significant ( P=0.34). Similarly, no significant differences were obtained if GSTT1 and/or GSTM1 null genotypes were analyzed in subgroups of control subjects and ovarian cancer patients between the ages of 20–40, 41–70 and 71–90 years and in individuals with a positive family history of neoplastic disease. GSTT1 and/or GSTM1 null genotypes were not significantly associated with the histologic type and grade or FIGO (International Federation of Gynecology and Obstetrics) stages of the ovarian carcinomas. In conclusion, GSTT1 and/or GSTM1 null genotypes are not markers for an increased risk of ovarian cancer.

A Multiplex Polymerase Chain Reaction Protocol for the Simultaneous Analysis of the GlutathioneS-Transferase GSTM1 and GSTT1 Polymorphisms

A Multiplex Polymerase Chain Reaction Protocol for the Simultaneous Analysis of the GlutathioneS-Transferase GSTM1 and GSTT1 Polymorphisms