Genus-specific Polymerase Chain Reaction Assay Research Articles

The development of genus-specific and species-specific real-time PCR assays for the authentication of Patagonian toothfish and Antarctic toothfish in commercial seafood products.

The substitution or mislabeling of toothfish is an issue of significant concern for seafood authorities; it also reduces the effectiveness of marine conservation and management programs for its over-exploitation and illegal trafficking, boosting the need for identification methods. Two species-specific real-time polymerase chain reaction (PCR) assays for the identification of Patagonian toothfish (Dissostichus eleginoides) and Antarctic toothfish (Dissostichus mawsoni) and a genus-specific real-time PCR assay for Dissostichus spp. identification were developed based on fragments of the 16S rRNA and COI (cytochrome c oxidase subunit I) genes. These methods were confirmed to be rapid, simple, and sensitive (absolute sensitivity of 0.0002 ng μL-1 and relative sensitivity of 0.1g kg-1 with good specificity). These methods can be applied to processed and commercial fish products. These approaches can be beneficial for protecting both consumers and producers from economic fraud and might also help protect toothfish from over-exploitation as well as combat illegal, unreported, and unregulated (IUU) fisheries. © 2021 Society of Chemical Industry.

Detection of Mycoplasma anatis, M. anseris, M. cloacale and Mycoplasma sp. 1220 in waterfowl using species-specific PCR assays.

Mycoplasma anatis, M. anseris, M. cloacale and M. sp. 1220 colonise geese and ducks, and could be associated with infections of avian respiratory and nervous systems, cause mild to severe inflammation of cloaca and genital tracts, and embryo lethality. Co-occurrence of these Mycoplasma species in waterfowl is frequently detected and the identification of these mycoplasmas to the species level at a regular microbiology laboratory is difficult due to their similar morphological, cultural and biochemical properties. Moreover, species differentiation is only possible based on the sequence analysis of the product of a genus-specific PCR assay. Therefore, the aim of the current study was to develop an effective and robust method for the identification of these species in avian clinical specimens. Polymerase chain reaction (PCR) assays using species-specific primers, which target housekeeping genes in order to identify these species, were designed in the present study. The developed PCR assays can precisely identify these four mycoplasmas to the species level directly from DNA samples extracted from clinical specimens, and no cross-amplification was observed among these species and with other well-known avian mycoplasmas. The average sensitivity of the assays was 101−102 genomic equivalents per reaction. These conventional PCR assays can be run simultaneously at the same PCR cycling program, and the species can be differentiated directly (without sequence analysis) by gel electrophoresis due to the specific sizes of the amplicons. In conclusion, the presented species-specific assays were found to be suitable for routine use at regular veterinary diagnostic laboratories and promote the rapid, simple and cost-effective differentiation of these waterfowl Mycoplasma species.

Serum based polymerase chain reaction and enzyme linked immunosorbent assays for diagnosis of bovine brucellosis

Brucellosis is one of the economically important diseases in India and diagnosis of brucellosis using single test is cumbersome due to variation in sensitivity and specificity among the different test. The present study was aimed to assess the suitability of serum as clinical specimen in molecular diagnosis and evaluate the serology and molecular assays as in diagnosis of bovine brucellosis. A total of 821 bovine sera samples were subjected to indirect Enzyme Linked Immunosorbent Assay (i-ELISA) and serum based Polymerase Chain Reaction (PCR) assay. On serology 6.70 per cent positivity of brucellosis were reported and on PCR assay, 47 and 29 sera samples were positive for bcsp 31 genus specific and IS711 species specific PCR assay respectively with per cent positivity of 5.72 and 3.53. In comparison between serology and molecular test, 44 samples were positive for both assays and 11 and 3 samples were positive for serology and molecular assays individually. This study suggests that serum sample can be utilised as the choice of clinical specimen for both PCR assay and i-ELISA will be a future choice for the diagnosis of bovine brucellosis.

Genus‐specific PCR assay for screening Arcobacter spp. in chicken meat

The number of emerging pathogenic species described within the genus Arcobacter has increased rapidly during the last few years. In this work a genus-specific polymerase chain reaction (PCR) assay was developed for detection of the species of Arcobacter most commonly associated with foods. The assay uses primers designed to amplify an 85 bp DNA fragment on the 16S rRNA gene and was applied to the detection of Arcobacter spp. in retail chicken meat. Primer specificity was tested against a panel of Arcobacter spp., related Campylobacter and Helicobacter spp. and other food bacteria. Arcobacter primers consistently and selectively amplified the expected DNA fragment in all tested Arcobacter spp. Bacterial control primers confirmed the presence of amplifiable DNA in the samples. The applicability of the PCR assay to food was validated through screening of fresh retail chicken samples for the presence of Arcobacter spp., with a result of 45% (23 out of 51) positive samples. The genus-specific PCR assay developed has the potential to be used as a quick and sensitive alternative method for the survey of Arcobacter contamination in meats.

Detection of Mycoplasma ovipneumoniae and M. arginini in Bighorn Sheep Using Enrichment Culture Coupled with Genus- and Species-Specific Polymerase Chain Reaction

Mycoplasma species are of interest as possible primary pathogens in the pneumonia complex of bighorn sheep (Ovis canadensis). Previous investigations have not commonly detected low frequencies of Mycoplasma spp. from free-ranging bighorn sheep, possibly due to the fastidious and slow growth of these organisms. We developed a culture protocol that employed an average initial 3-day enrichment culture in liquid Hayflick broth in a CO(2)-enhanced atmosphere. The broth was plated to solid Hayflick medium and the cultures observed for growth for up to 30 days. Polymerase chain reaction (PCR) was performed on DNA isolated from the enrichment broth and on isolates obtained from culture using Mycoplasma genus-specific PCR assays and species-specific PCR assays for M. arginini and M. ovipneumoniae. Some cultures that grew on Hayflick plates were picked as single colonies but were mixed because two organisms may grow together and appear as a single colony. Culture and PCR tests produced similar results for M. arginini, but for M. ovipneumoniae, culture alone was less accurate than PCR. Use of genus-specific primers also may allow detection of other species in samples negative for M. arginini and M. ovipneumoniae. Two methods of transport from field to laboratory (Port-a-Cul™ tubes, cryoprotectant in liquid N(2) and Fisher Transport System) gave similar results under our study conditions.

Multiplex polymerase chain reaction (PCR) assays for the detection of Enterobacteriaceae in clinical samples

The accurate and rapid identification of bacteria in the enteric tract is necessary for early treatment. In this study, we describeD a novel system which consists of a multiplex polymerase chain reaction (PCR) to simultaneously identify a group of six Enterobacteriaceae members including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter spp., Enterobacter cloacae and Salmonella typhi. Genus and species specific primers were designed for this group of pathogens and conventional multiplex PCR and SYBR green based real time PCR assays were performed to detect these pathogens. All the samples were analysed with a eubacterial real-time PCR assay that enables detection of bacterial DNA and then detection of the organisms was determined using genus and species specific PCR assays. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. Their PCR results matched with the conventional culture identifications. The conventional and SYBR green real time multiplex PCR assays takes only 3 h to be performed and has the potential to replace the conventional culture technique and thus can speed up the treatment process. This technique has the potential to be a valuable diagnostic tool for simultaneous identification of E. coli, K. pneumoniae, P. mirabilis, Citrobacter spp., E. cloacae and S. typhi.

Prevalence of Bordetella pertussis and Bordetella parapertussis in Infants Presenting to the Emergency Department with Bronchiolitis

The clinical presentation of Bordetella pertussis can overlap with that of respiratory syncytial virus (RSV), and coinfection does occur, but management differs. The prevalence of B. pertussis is < 2% among Emergency Department (ED) patients with bronchiolitis. Our secondary hypothesis was that the prevalence of Bordetella parapertussis is also < 2% among these patients. Nasal washings were obtained from children up to 18 months of age (inclusive) who presented to a county hospital ED with a clinical diagnosis of bronchiolitis. These washings were frozen to -70°C before testing for B. pertussis and B. parapertussis using species-specific real-time polymerase chain reaction (PCR) assays. The assays were optimized to target conserved regions within a complement gene and the CarB gene, respectively. A Bordetella spp. genus-specific real-time PCR assay was designed to detect the Bhur gene of B. pertussis, B. parapertussis, and B. bronchiseptica. RSV antigen detection was also performed. There were 227 patients enrolled. After exclusions, 204 remained in the analysis. RSV antigen testing was positive in 109/186 (59%) of the patients in whom it was performed. All samples were tested for B. pertussis. B. parapertussis testing could not be completed on 23 samples. No cases (0/204; 95% confidence interval [CI] 0-1.8%) tested positive for B. pertussis or B. parapertussis (0/181; 95% CI 0-2%). The prevalence of B. pertussis in children presenting to the ED with bronchiolitis was < 2%.

Development of a genus-specific PCR assay for the molecular detection, confirmation and identification of Fusobacterium spp

A genus-specific polymerase chain reaction (PCR)-based assay is developed for the detection and identification of clinically relevant Fusobacterium species, including F. nucleatum and F. necrophorum. Two 16S ribosomal DNA (rDNA) primers, FUSO1 (forward primer: 5’-GAG AGA GCT TTG CGT CC-3’ [17-mer]) and FUSO 2 (reverse primer: 5’-TGG GCG CTG AGG TTC GAC -3’ [18-mer]) are designed to target conserved regions of the 16S rDNA gene for Fusobacterium spp. Subsequent proof-of-principle studies employing this assay detected Fusobacterium spp. in the faeces of eight (10%) out of 80 patients with suspected gastrointestinal infection. This assay may be used for the genus-specific detection of Fusobacterium spp. from clinical specimens and for subsequent species identification.

Health Assessment of the Guam Rail (Gallirallus owstoni) Population in the Guam Rail Recovery Program

ABSTRACT A health assessment of the captive island population of Guam rail (Gallirallus owstoni) included a review of pathology records, diet analysis, and physical examination and diagnostic findings in prerelease and captive breeding populations (n = 100 and n = 55, respectively). The general health of domestic chickens (n = 50) on Guam and Rota was also assessed. For rails, diagnostic tests included a complete blood count, plasma biochemical analysis, plasma protein electrophoresis, genus-specific plasma enzyme-linked immunoassay (ELISA) for Mycobacterium avium complex (MAC) antigen, arboviral serologic testing, culture for enteric pathogens (Salmonella and Campylobacter species) and Mycobacterium species, fecal examination for parasites, and genus-specific polymerase chain reaction assay for fecal MAC antigen. For chickens, diagnostic tests included arboviral serologic tests and fecal culture for enteric pathogens and Mycobacterium species. Results of testing in the rails were negative for arbov...

Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids

An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the identification of six Candida species and four Aspergillus species in pure cultures of clinical isolates, as well as in bronchoalveolar lavage (BAL) fluid samples from 42 patients with various underlying diseases. The results were compared with the results obtained with conventional routine identification methods as well as with a commercial enzyme-linked immunosorbent assay (ELISA) galactomannan detection assay and an Aspergillus-specific PCR. No discrepancies between the PCR-LiPA system and routine methods were found for pure cultures of Candida and Aspergillus species except in the case of Aspergillus versicolor. In BAL fluid samples in which Candida species were cultured, the PCR-LiPA system identified more species than did the routine methods. When routine analyses of patient samples were supplemented by adding data obtained by repurifying and re-identifying cultures and by taking isolates obtained from other body sites into account, the results agreed with PCR-LiPA system results in 81% of the cases (34/42). Most of the remaining discrepancies (6/8) involved cases in which such supplementary data were not available. In BAL fluid samples from which A. fumigatus was cultured, the agreement between the PCR-LiPA system and the routine methods was low. Only 2 of 11 BAL samples shown to contain A. fumigatus in ELISA and genus-specific PCR assays were positive in PCR-LiPA system. The PCR-LiPA system enables the simultaneous detection and identification of different fungal species present in pure or mixed populations within 6 h in a single assay. Optimization is required, however, before it is useful as a diagnostic tool in the clinical microbiology laboratory.

Evaluation of a polymerase chain reaction reverse hybridization line probe assay for the detection and identification of medically important fungi in bronchoalveolar lavage fluids.

An assay system in which polymerase chain reaction (PCR) amplification of the ITS-1 region of ribosomal DNA (rDNA) is combined with a reverse-hybridization line probe assay (LiPA) was used for the identification of six Candida species and four Aspergillus species in pure cultures of clinical isolates, as well as in bronchoalveolar lavage (BAL) fluid samples from 42 patients with various underlying diseases. The results were compared with the results obtained with conventional routine identification methods as well as with a commercial enzyme-linked immunosorbent assay (ELISA) galactomannan detection assay and an Aspergillus-specific PCR. No discrepancies between the PCR-LiPA system and routine methods were found for pure cultures of Candida and Aspergillus species except in the case of Aspergillus versicolor. In BAL fluid samples in which Candida species were cultured, the PCR-LiPA system identified more species than did the routine methods. When routine analyses of patient samples were supplemented by adding data obtained by repurifying and re-identifying cultures and by taking isolates obtained from other body sites into account, the results agreed with PCR-LiPA system results in 81% of the cases (34/42). Most of the remaining discrepancies (6/8) involved cases in which such supplementary data were not available. In BAL fluid samples from which A. fumigatus was cultured, the agreement between the PCR-LiPA system and the routine methods was low. Only 2 of 11 BAL samples shown to contain A. fumigatus in ELISA and genus-specific PCR assays were positive in PCR-LiPA system. The PCR-LiPA system enables the simultaneous detection and identification of different fungal species present in pure or mixed populations within 6 h in a single assay. Optimization is required, however, before it is useful as a diagnostic tool in the clinical microbiology laboratory.

Failure to isolate Helicobacter pylori from stray cats indicates that H. pylori in cats may be an anthroponosis--an animal infection with a human pathogen.

The recent isolation of Helicobacter pylori from cats obtained from a commercial supplier has potentially important public health implications. The present study investigated whether H. pylori infection was common in stray cats. Twenty-five cats were examined for the presence of H. pylori by histological examination, culture and two polymerase chain reaction (PCR) assays. Histologically, the gastric biopsy specimens from all cats showed large spiral organisms typical of H. felis and not H. pylori. Samples from 23 cats yielded bacterial growth and two had no growth. Colonies grossly similar to H. pylori were tested for catalase, oxidase, urease and Gram's stain reactions. None was H. pylori. All samples tested as positive by the Helicobacter 16S rRNA genus-specific PCR assay and only six cats and a mouse stomach infected with H. heilmannii gave positive results with the adhesin subunit A (hpaA)-specific PCR assay, which is consistent with either H. pylori or H. heilmannii. The helicobacters identified in these samples by PCR were not cultivable and hence were probably H. heilmannii. H. pylori infection is uncommon in stray cats and owning pet cats should not be a threat to public health in relation to H. pylori infection.