Polymerase Chain Reaction Primer Sets Research Articles

Clinical, molecular and cytogenetic analysis of 46, XX testicular disorder of sex development with SRY-positive.

BackgroundTo review the possible mechanisms proposed to explain the etiology of 46, XX sex reversal by investigating the clinical characteristics and their relationships with chromosomal karyotype and the SRY(sex-determining region Y)gene.MethodsFive untreated 46, XX patients with SRY-positive were referred for infertility. Clinical data were collected, and Karyotype analysis of G-banding in lymphocytes and Fluorescence in situ hybridization (FISH) were performed. Genomic DNA from peripheral blood of the patients using QIAamp DNA Blood Kits was extracted. The three discrete regions, AZFa, AZFb and AZFc, located on the long arm of the Y chromosome, were performed by multiplex PCRs(Polymerase Chain Reaction) amplification. The set of PCR primers for the diagnosis of microdeletion of the AZFa, AZFb and AZFc region included: sY84, sY86, sY127, sY134, sY254, sY255, SRY and ZFX/ZFY.ResultsOur five patients had a lower body height. Physical examination revealed that their testes were small in volume, soft in texture and normal penis. Semen analyses showed azoospermia. All patients had a higher follicle-stimulating hormone(FSH), Luteinizing Hormone(LH) level, lower free testosterone, testosterone level and normal Estradiol, Prolactin level. Karyotype analysis of all patients confirmed 46, XX karyotype, and FISH analysis showed that SRY gene were positive and translocated to Xp. Molecular analysis revealed that the SRY gene were present, and the AZFa, AZFb and AZFc region were absent.ConclusionsThis study adds cases on the five new 46, XX male individuals with SRY-positive and further verifies the view that the presence of SRY gene and the absence of major regions in Y chromosome should lead to the expectance of a completely masculinised phenotype, abnormal hormone levels and infertility.

Polymerase Chain Reaction molecular diagnostic technology for monitoring chronic osteomyelitis.

BackgroundOsteomyelitis is a devastating condition whose treatment relies on the detection of bacteria. The current standard of microbiology culture may not be adequate. Molecular biology based diagnostic procedures for detecting bacteria in orthopaedic infections was previously established, but has not been applied to the setting of chronic osteomyelitis. We aim to determine the applicability of molecular diagnostic procedures for monitoring chronic osteomyelitis, and to evaluate if these procedures are superior to standard culture methods of osteomyelitis detection.MethodsA rabbit experimental model of chronic osteomyelitis was used; infection was induced in the proximal, medial aspect of the tibia with Staphylococcus aureus at titers ranging from 1 × 102 to 1 × 106 colony forming units. At 28 days post-infection, animals were sacrificed, and the tibias were examined radiographically, harvested, and assayed for the presence of bacteria. Two bacterial detection methods were used: (1) standard microbiological culturing, and (2) polymerase chain reaction (PCR) based diagnostic method to detect bacterial genomic DNA.ResultsThe molecular diagnostic method was highly sensitive and accurate, and detected low titer infections that were undetected by radiographic and microbiological methods. By using two sets of PCR primers, one for a universal bacterial gene (16S rRNA) and one for a species-specific gene (nuc), the molecular protocol allowed both the detection and speciation of the bacterial infection.ConclusionsThe use of the PCR-based method was effective for high-sensitivity detection and identification of bacteria associated with chronic osteomyelitis in a rabbit model. Our findings illustrate the applicability of PCR for monitoring chronic osteomyelitis, which may be useful for improved detection of osteomyelitis organisms in humans.

Molecular detection of Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Burkholderia glumae in infected rice seeds and leaves

The polymerase chain reaction (PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an AvrRxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer (ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10fgμL−1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.

English

Megasphaera micronuciformis is an anaerobic microbe isolated from humans. However, since the microbe is strictly anaerobic, its cultivation requires complicated facilities, making detection costly. For rapid, inexpensive detection and identification of M. micronuciformis in the clinical setting, a new technique is necessary. This study aimed to develop a species-specific PCR primer set for the detection of M. micronuciformis. A ribosomal DNA-specific PCR primer Mm2F was designed for M. micronuciformis. Analytical specificity data showed that the PCR primer set Mm2F/Mega-X produced amplicons from M. micronuciformis but not from the other species tested, including 4 Megasphaera species and representative related species. Of the 52 oral samples from Japanese subjects evaluated in our study, 71% were positive for M. micronuciformis, suggesting the likelihood that M. micronuciformis is widely distributed in the oral cavity of the Japanese population.    Key words: Megasphaera micronuciformis, specific polymerase chain reaction (PCR), Mm2F, oral bacteria.

Development of a polymerase chain reaction applicable to rapid and sensitive detection of Clonorchis sinensis eggs in human stool samples

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity.

Analytical Validation of a Practical Molecular Assay Prognostic of Survival in Nonsquamous Non–Small Cell Lung Cancer

A molecular assay prognostic of survival in resected nonsquamous non-small cell lung cancer designed to meet the need for improved risk stratification in early-stage disease has recently been described. This assay measures the expression levels of 14 genes using RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. The assay underwent blinded clinical validation in 2 large international cohorts involving approximately 1500 patients; the analytical precision and reproducibility of this assay, however, have not yet been reported. For each of the 14 TaqMan quantitative polymerase chain reaction (PCR) primer and probe sets used in the molecular prognostic assay, the linear range, PCR efficiency, limits of blank, limits of quantitation, and quantitative bias were determined using serial dilutions of pooled RNA extracted from FFPE samples. The reproducibility of the entire molecular assay was determined by performing repeat testing of FFPE samples over multiple days. The linear range of individual quantitative TaqMan PCR primer and probe sets was between 2(10)- and 2(15)-fold input RNA. The median C(T) of the quantitative PCR primer and probe sets at 10 ng of input RNA was 24.3; the median efficiency was 91.2%. The median quantitative bias across all quantitative PCR primer and probe sets was 0.75% (range, 0.32% to 1.32%). In repeat testing, the mean SD of the risk score (scaled from 1 to 100) was 2.18, with a mean coefficient of variation of 0.08. The molecular prognostic assay presented in this study demonstrates high precision and reproducibility, validating its clinical utility as a reliable prognostic tool that can contribute to the management of patients with early-stage disease.

Development of PCR Primers to Identify Fusarium oxysporum f. sp. fragariae.

Fusarium oxysporum f. sp. fragariae is a fungal pathogen causing Fusarium wilt on strawberry. Polymerase chain reaction (PCR) primers that can discriminate F. oxysporum f. sp. fragariae from nonpathogenic F. oxysporum would greatly assist pathogen identification. In order to develop a molecular diagnostic tool for this pathogen, transposable elements in the pathogen were characterized and used for designing a specific set of PCR primers. Portions of the transposable elements Fot3, Han, Hop, Hornet1, and Skippy were detected in all 33 strains of F. oxysporum f. sp. fragariae tested by PCR, whereas Foxy was detected in 32 strains and Impala sequences were detected in 30 strains. Two types of sequences were detected for Hop, two types for Impala, and three types for Skippy. The genomic region between Han and Skippy was amplified by an inter-retrotransposon amplified polymorphism technique, and PCR primers (FofraF and FofraR) to specifically identify F. oxysporum f. sp. fragariae were designed from this region. The developed PCR primers discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and five other formae speciales. Conidia of F. oxysporum f. sp. fragariae could be detected in brown lowland-type soil by PCR using the primers. After preculturing the soil sample on FoG2 medium, 1 × 102 conidia/g of soil could be detected; without preculturing, 1 × 103 conidia/g of soil were detected.

Strain-Specific Identification ofBifidobacterium bifidumOLB6378 by PCR

The aim of the present study was to develop a strain-specific polymerase chain reaction (PCR) primer set for the detection of Bifidobacterium bifidum OLB6378 (OLB6378) that can serve as suitable probiotics for infants. The random amplified polymorphic DNA (RAPD)-PCR technique was used to obtain OLB6378-specific PCR products. One OLB6378-specific RAPD-PCR product was obtained after testing 97 RAPD primers, and was sequenced. Thirteen PCR primer sets were designed from the sequence. One PCR primer set was found to amplify one PCR product when genomic DNA of OLB6378 was used as template. The primer set did not amplify any PCR product when the other genomic DNA was used as template. The primer set was tested with 47 strains of B. bifidum and 20 strains of the other Bifidobacterium species. As a result, we developed an OLB6378-specific primer set, one that should be useful not only for the detection of OLB6378 but also for the quantification of OLB6378.

Characterization of Four New Mouse Cytochrome P450 Enzymes of the CYP2J Subfamily

The cytochrome P450 superfamily encompasses a diverse group of enzymes that catalyze the oxidation of various substrates. The mouse CYP2J subfamily includes members that have wide tissue distribution and are active in the metabolism of arachidonic acid (AA), linoleic acid (LA), and other lipids and xenobiotics. The mouse Cyp2j locus contains seven genes and three pseudogenes located in a contiguous 0.62 megabase cluster on chromosome 4. We describe four new mouse CYP2J isoforms (designated CYP2J8, CYP2J11, CYP2J12, and CYP2J13). The four cDNAs contain open reading frames that encode polypeptides with 62-84% identity with the three previously identified mouse CYP2Js. All four new CYP2J proteins were expressed in Sf21 insect cells. Each recombinant protein metabolized AA and LA to epoxides and hydroxy derivatives. Specific antibodies, mRNA probes, and polymerase chain reaction primer sets were developed for each mouse CYP2J to examine their tissue distribution. CYP2J8 transcripts were found in the kidney, liver, and brain, and protein expression was confirmed in the kidney and brain (neuropil). CYP2J11 transcripts were most abundant in the kidney and heart, with protein detected primarily in the kidney (proximal convoluted tubules), liver, and heart (cardiomyocytes). CYP2J12 transcripts were prominently present in the brain, and CYP2J13 transcripts were detected in multiple tissues, with the highest expression in the kidney. CYP2J12 and CYP2J13 protein expression could not be determined because the antibodies developed were not immunospecific. We conclude that the four new CYP2J isoforms might be involved in the metabolism of AA and LA to bioactive lipids in mouse hepatic and extrahepatic tissues.

Additional sequence characterization of NIST SRM 2391c: PCR-Based DNA Profiling Standard

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was designed for use in the standardization of forensic and paternity quality assurance procedures for fragment-based typing short tandem repeat (STR) alleles generated by the polymerase chain reaction (PCR). Certified genotypes of the 6 components A–F were assigned for 24 autosomal and 17 Y-STR markers plus Amelogenin using concordance testing between commercial kits. Selected Sanger sequencing characterization was performed for the alleles of 11 STR markers when only one PCR primer set was available for fragment-based typing. The goal is to characterize the remaining 30 STR loci in components A–C by Sanger sequencing methods for the STR repeat regions and adjacent flanking regions. Additional characterization of the SRM is intended to support the emerging interest in next-generation sequencing technologies for forensic typing applications. Sanger methods have detected underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. The sequenced regions include the commercial or known PCR binding sites commonly implemented in fragment-based typing.

Detection of Vibrio cholerae and Vibrio vulnificus by duplex PCR specific to the groEL gene

Vibriocholerae and V. vulnificus are of major concern due to their effect on public health throughout the world. It is therefore imperative to identify a gene and method that are suitable for the accurate species-specific detection of these two species. A duplex polymerase chain reaction (PCR) assay was developed using two sets of primers targeting the groEL gene for the accurate simultaneous detection of V. cholerae and V. vulnificus. The nucleotide sequence of the groEL gene was compared with the sequences of other Vibrio and non-Vibrio species. The specificity of two primer sets for duplex PCR was checked using 24 Vibrio and 8 non-Vibrio species. The primer sets were found to be specific for these two species and could detect both of the target bacterial species without any ambiguity, even when comparing closely related species. For both species, the detection limit was 100 pg of purified genomic DNA. The duplex PCR showed high specificity and sensitivity for each species and was sufficient for the detection of V. cholerae and V. vulnificus from artificially infected shellfish tissue, flounder, and even inoculated seawater. This method is simple and cost-effective, and can be utilized for the simultaneous detection of both species, thus representing an effective tool for both epidemiologist and ecologist.

Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R

Salmonella enterica serovar Gallinarum biovar Gallinarum causes fowl typhoid in chickens and has been of economic importance to the chicken industry in many countries. The biovar Gallinarum live vaccine strain 9R (SG 9R) has been used to control fowl typhoid in many areas where the disease is endemic. Therefore, a definitive diagnosis of this disease may require differentiation of wild-type field isolates of biovar Gallinarum from the live vaccine strain SG 9R. Here, we report the development of a triplex polymerase chain reaction (PCR) assay to differentially identify serovar Gallinarum biovars Gallinarum and Pullorum and SG 9R. Sequences specific to SG 9R, which are absent or highly divergent in the fully sequenced biovar Gallinarum strain 287/91, were identified by constructing the suppression subtractive hybridization (SSH) library. A total of 565 nonredundant inserts were obtained from successfully sequenced SSH clones (718 clones). Sequences of 14 inserts were unique to SG 9R, but single nucleotide polymorphisms (SNPs) found in another insert (9R22C9) were more useful for strain discrimination. A new PCR primer set was designed to target SNP regions of the insert and was integrated into a duplex PCR assay developed previously (Kang et al., 2011). Boiled lysates of 138 reference and field strains of Salmonella and other related Gram-negative bacteria were tested to validate the triplex PCR assay. All strains of biovars Gallinarum (n=53) and Pullorum (n=21) and SG 9R (n=7) tested were differentially identified, whereas the other strains (n=57) were PCR negative. This triplex PCR assay will be very useful for rapid differential diagnoses of fowl typhoid and pullorum disease in veterinary laboratories.

Value of Merkel cell Polyomavirus DNA Detection in Routine Pathology

To the Editor: With great interest, we studied the recently published article from Duncavage et al1 “Merkel Cell Polyomavirus-A Specific Marker for Merkel Cell Carcinoma in Histologically Similar Tumors”. We agree that molecular pathologic investigation for Merkel cell polyomavirus (MCPyV) of tumors, which are histologically similar to Merkel cell carcinoma (MCC) is a very interesting and important issue. It is beyond debate that analysis of MCPyV prevalence in other skin conditions than MCC is necessary to get insight in the relevance of MCPyV infection. Our group is intensively working in this field, and we discuss the value of MCPyV detection as a diagnostic tool in routine histopathology in the light of the latest literature. Duncavage et al1 screened 74 cases of visceral high-grade neuroendocrine tumors (which are histologically similar to MCC) for MCPyV DNA by means of polymerase chain reaction (PCR). They detected MCPyV DNA just in 1 case (1.4%). Interestingly this case is, “upon detailed review, actually a misclassified case of metastatic MCC”, displaying the problem of distinguishing these entities. Therefore they suggest PCR-based testing for MCPyV DNA as helpful and specific marker in differentiating those histologically similar tumors. Furthermore, they report and discuss frequent absence of MCPyV DNA in several cutaneous and extracutaneous neoplasms.1 We agree with Duncavage et al1 that detection of MCPyV DNA is highly dependent upon a number of different methodical parameters such as tissue fixation, methods of DNA extraction and specific sequence detection, such as PCR method, selection of PCR primer sets, and last but not least dependent upon the amount of available tissue. Among others, Duncavage et al1 investigated 16 small cell carcinomas of the lung reporting absence of MCPyV DNA. We analyzed in a prior study, 30 small cell carcinomas of the lung and detected MCPyV DNA in 2 of 30 patients (7.5%), in 3 of 35 samples (2 of these 3 samples were from the same patient), respectively.2 Furthermore, there is enough evidence that MCPyV DNA is a common and frequent finding in tissues of immunosuppressed and immunocompetent individuals as well. MCPyV DNA was found in several benign, semimalignant and malignant skin tumors as well as in inflammatory skin diseases and clinical healthy skin from non-MCC patients.3-12Table 1 summarizes an incomplete selection of results from various research groups analyzing MCPyV DNA prevalence in different entities. For example, prevalence of MCPyV DNA in basal cell carcinomas was investigated from 4 independent research groups and taken together MCPyV DNA was detected in 66 of 221 samples (29.9%), ranging from 0% to 72%.3,5,6,8 We think, this situation nicely shows that MCPyV DNA analysis is most likely dependent on various before mentioned technical parameters, resulting in variable MCPyV DNA detection rates. Nevertheless, almost any of these studies point to the fact that MCPyV is ubiquitously present, even detectable in healthy and non-MCC tissue. Of special interest is that various groups reported significant differences in MCPyV viral load with usually higher copy numbers in MCC tissues and lower ones in non-MCC tissues, representing a possible parameter for differentiation.5,6,9 However, one must consider that most routine pathology laboratories use standard PCR-based methods for MCPyV detection as a qualitative test without analyzing quantitative viral load.TABLE 1: Prevalence of MCPyV DNA in Different Entities Analyzed by PCR-Based MethodsIn summary, latest reports proved that MCPyV DNA is abundantly detectable in tissues of various entities including clinical healthy skin (Table 1). Therefore, a qualitative proof of MCPyV DNA in a tissue sample is by far not specific for MCC. To us, the value of an ancillary MCPyV DNA-analysis in the differential diagnosis of MCC and similar tumors is questionable, and results have to be interpreted in the context of the recent literature suggesting a high prevalence of MCPyV. ACKNOWLEDGMENTS This study was in part supported by the Dr. H. Legerlotz, the R. Bartling and the M. Lackas Foundation. Christian Andres, MD* Benedetta Belloni, MD* *Department of Dermatology and Allergy, Ludwig-Maximilians-Universität München, Munich, Germany Michael J. Flaig, MD† †Department of Dermatology and Allergy Biederstein, Technische Universität München, Munich, Germany

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species

The gram-positive bacterial genus Lactococcus has been taxonomically classified into seven species (Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis and Lactococcus fujiensis). This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of the seven lactococcal species, as well as to differentiate the two industrially important dairy subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris. A multiplex PCR primer set was designed based on the nucleotide sequences of the 16S rRNA gene of the seven lactococcal species. The specificity of the established one-step multiplex PCR scheme was verified using more than 200 bacterial strains, in which a complete sequence match was confirmed by partial sequencing of their 16S rRNA gene. The one-step multiplex PCR enables the identification and speciation of bacterial strains belonging to the genus Lactococcus and the differentiation of strains of L. lactis subsp. lactis and L. lactis subsp. cremoris. This work provides an efficient method for identification of lactococcal strains of industrial importance.

A protocol for a single-cell PCR of diatoms from fixed samples: method validation using Ditylum brightwellii (T. West) Grunow

Many major advances in macro-organismal biology, population genetics and evolutionary biology may be attributed to the development of rapid and inexpensive molecular methodology. Here, a simple protocol is presented for a polymerase chain reaction (PCR) of alcohol-preserved single diatom cells that facilitate studies requiring a large number of individuals, inclusive of archived and uncultivable specimens. Our approach integrates cell isolation and PCR amplification of two molecular markers, as well as post-PCR identification by scanning electron microscopy, thereby bridging the gap between classical and molecular diatom biology. The amplification of two genes was performed in a two-step protocol. By applying multiple sets of PCR primers to a single diatom cell, an internal fragment of the large subunit of the ribulose-1,5-bisphosphate carboxylase gene (RuBisCO, rbcL) was successfully amplified and sequenced, as well as the internal transcribed spacer (ITS) region in 20% of the same, preserved cells derived from natural phytoplankton. Multiple-gene recovery from a single-cell source requires further optimization, likely specific for the taxa of interest. Single marker recovery (from first-step amplification to sequencing) from natural, fixed single-cell samples was more successful with overall success rate of ∼31% for ITS and 67% for rbcL, respectively. Admittedly it is 50–70% lower than rates for a culture-pelleted DNA source (68% for ITS and 100% for rbcL) using current standard methods of sequence recovery in diatom studies, but our method is much less labor- and cost-intensive. This approach was applied to the diatom Ditylum brightwellii to assess the quality of recovered sequences. The phred quality scores of our sequences derived from single-cell material were similar to phred scores of our own, published sequences obtained from cultures grown in our laboratory.

Development of Prevotella nigrescens-specific PCR primers based on the nucleotide sequence of a Pn23 DNA probe

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop P. nigrescens-specific polymerase chain reaction (PCR) primers. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. Interestingly, the two sets of PCR primers, Pn23-F6/Pn23-R6 and Pn23-F7/Pn23-R7, had strain-specificity for P. nigrescens ATCC 33563. The detection limits of the four primer sets were 40 or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.

A quantitative PCR method to quantify ruminant DNA in porcine crude heparin.

Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550–554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3–3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity.

Detection ofPseudomonas syringaepv.actinidiaeusing polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

Several published polymerase chain reaction (PCR) primers to identifyPseudomonas syringaepv.actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains ofP. syringaepv.actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen,P. syringaepv.theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections asP. syringaepv.actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB,acnB,rpoD,pgiandcts) showed that none of these six strains was phylogenetically similar toP. syringaepv.actinidiae.In contrast to theP. syringaepv.actinidiaetype strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified asP. syringaepv.actinidiae. It was not possible to distinguishP. syringaepv.actinidiaefrom the phylogenetically similarP. syringaepv.theaeusing the ITS,gyrB,acnB,rpoD,pgiorctsgene regions to design PCR primers. BecauseP. syringaepv.theaeis unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.

Use of PCR primers and probes based on the 23S rRNA and internal transcription spacer (ITS) gene sequence for the detection and enumerization of Lactobacillus acidophilus and Lactobacillus plantarum in feed supplements

Novel polymerase chain reaction (PCR) primers designed from the 16S–23S internal transcription spacer (ITS) rRNA and 23S rRNA genes, respectively, were used for the specific detection of Lactobacillus acidophilus and Lactobacillus plantarum. Molecular weights of the PCR products were 221 and 599 bp, respectively. Strains of L. acidophilus and L. plantarum obtained from the culture center, dairy products, infant stool and other samples, could be identified with these PCR primers. DNAs from other lactic acid bacteria (LAB) species including strains of Lactobacillus pentosus which was closely related to L. plantarum, and bacteria species other than LAB, would not generate the false positive results. When this PCR primer set was used for the detection of L. acidophilus and L. plantarum in feed supplement or feed starter samples, reliable results were obtained. Furthermore, when these L. acidophilus or L. plantarum specific primers were used as DNA probes for the colony hybridization of L. acidophilus and L. plantarum, the viable cells of these LAB species in culture and feed supplements or starter products could be identified and enumerized. The method described here thus offers a rapid and economic way to inspect and assure the quality of the feed supplements or fermentation starters.

A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments

Published polymerase chain reaction primer sets for detecting the genes encoding 16S rRNA gene and hydrazine oxidoreductase (hzo) in anammox bacteria were compared by using the same coastal marine sediment samples. While four previously reported primer sets developed to detect the 16S rRNA gene showed varying specificities between 12% and 77%, an optimized primer combination resulted in up to 98% specificity, and the recovered anammox 16S rRNA gene sequences were >95% sequence identical to published sequences from anammox bacteria in the Candidatus "Scalindua" group. Furthermore, four primer sets used in detecting the hzo gene of anammox bacteria were highly specific (up to 92%) and efficient, and the newly designed primer set in this study amplified longer hzo gene segments suitable for phylogenetic analysis. The optimized primer set for the 16S rRNA gene and the newly designed primer set for the hzo gene were successfully applied to identify anammox bacteria from marine sediments of aquaculture zone, coastal wetland, and deep ocean where the three ecosystems form a gradient of anthropogenic impact. Results indicated a broad distribution of anammox bacteria with high niche-specific community structure within each marine ecosystem.