Real-Time PCR for Detection and Identification of Anguina funesta, A. agrostis, A. tritici, and A. pacificae

A number of seed, leaf, and stem gall nematodes are of significance to the forage and landscape grass and livestock industries. In North America, the bentgrass nematode, Anguina agrostis, reduces seed production on Agrostis tenuis and several other grass species. Anguina funesta is a seed-gall nematode that is most significant for its association with the toxigenic bacteria Rathayibacter toxicus. The wheat seed gall nematode A. tritici causes significant damage to wheat and other cereals; although it has been found in many countries worldwide, it has not been detected in the United States since 1975. Molecular methods based upon sequence variation in the ribosomal internal spacer region are useful for accurate identification of Anguina spp. Described herein are new species-specific primers and TaqMan probes for real-time polymerase chain reaction (PCR) identification of A. agrostis, A. funesta, A. tritici, and A. pacificae. Primer and probe combinations were each specific for the intended species and were sensitive enough to detect as few as 1.25 copies of nematode ribosomal DNA. PCR was also specific and sensitive in duplex assays that included genus-specific internal control primers as well as species-specific primers and probes. These standardized real-time PCR protocols should facilitate fast and accurate identification of Anguina spp. by diagnostic laboratories.

Download Full-text

Developing, Modifying, and Validating a TaqMan Real-Time PCR Technique for Accurate Identification of Leishmania Parasites Causing Most Leishmaniasis in Iran

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.731595 ◽

2021 ◽

Vol 11 ◽

Author(s):

Reza Fotouhi-Ardakani ◽

Seyedeh Maryam Ghafari ◽

Paul Donald Ready ◽

Parviz Parvizi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Major ◽

Taqman Probe ◽

Amino Acid Permease ◽

Specific Primers ◽

Accurate Identification ◽

Parasitic Load ◽

Species Specific

Many laboratory methods are used to diagnose leishmaniasis because it is characterized by varied symptoms and caused by different Leishmania species. A quantitative real-time PCR method based on a TaqMan probe was developed and modified for accurate identification of human cutaneous leishmaniasis (caused by Leishmania major or Leishmania tropica) from endemic areas of Iran. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were considered. Six new sets of species-specific primers and probes were designed. A total of 123 samples were examined and employed to evaluate and validate real-time PCR. According to parasitic load of the genesig®Leishmania Advanced Standard Kit, a serial dilution of purified plasmid (2–2×107 copies/reaction) was prepared under the same conditions for both genes. Specific primers and probes were able to detect three and six parasite copies in AAP3 and COII genes, respectively, and were able to detect three copies of parasites for L. major and L. tropica. The sensitivities of the reference kit and our method were 98.7 and 98.1%, respectively, and specificity was 100% for detecting parasite genomes in all assays. Designed primers and probes performed well in terms of efficiency and regression coefficient. For AAP3 and COII genes, respectively, the linear log range was 7 and the correlation coefficient (R2) was 0.749 and 0.996 for the reference kit using the standard generated curve and 0.98 and 0.96 with serial dilutions of parasite DNA. This research detected L. major and L. tropica definitely and opens the horizon for the other scientists in the multiplex reactions in designing and optimization of the conditions in silico and in vivo.

Download Full-text

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.70.1.167-173.2004 ◽

2004 ◽

Vol 70 (1) ◽

pp. 167-173 ◽

Cited By ~ 309

Author(s):

Takahiro Matsuki ◽

Koichi Watanabe ◽

Junji Fujimoto ◽

Yukiko Kado ◽

Toshihiko Takada ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Real Time Pcr ◽

Intestinal Flora ◽

Healthy Adults ◽

Pcr Detection ◽

Distribution Analysis ◽

Specific Primers ◽

Cell Counts ◽

Species Specific

ABSTRACT A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 106 to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >106 cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.

Download Full-text

FAST DETECTION AND IDENTIFICATION OF ICU-RELEVANT BACTERIA BY REAL-TIME PCR AND SPECIES-SPECIFIC PROBES

Shock ◽

10.1097/00024382-200403001-00427 ◽

2004 ◽

Vol 21 (Supplement) ◽

pp. 107

Author(s):

S. Klaschik ◽

L E Lehmann ◽

A. Hoeft ◽

F. Stuber

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Fast Detection ◽

Detection And Identification ◽

Species Specific

Download Full-text

Development of Primers and Probes for Genus and Species Specific Detection of ‘Candidatus Liberibacter Species’ by Real-Time PCR

Plant Disease ◽

10.1094/pdis-12-12-1174-re ◽

2013 ◽

Vol 97 (9) ◽

pp. 1235-1243 ◽

Cited By ~ 13

Author(s):

G. Ananthakrishnan ◽

N. Choudhary ◽

Avijit Roy ◽

V. G. Sengoda ◽

E. Postnikova ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Diagnostic Methods ◽

Rrna Gene ◽

Single Pair ◽

Degenerate Primers ◽

Specific Primers ◽

Genus Level ◽

Candidatus Liberibacter ◽

Species Specific

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different ‘Candidatus Liberibacter species’, ‘Ca. Liberibacter asiaticus’, ‘Ca. Liberibacter americanus’, and ‘Ca. Liberibacter africanus’, which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of ‘Ca. Liberibacter’ at the genus level, providing increased convenience. Recent molecular analyses of ‘Ca. Liberibacter species’ and other bacteria suggest that the rpoB gene (encoding the β-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus ‘Ca. Liberibacter species’, enabling detection of ‘Ca. Liberibacter’ at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected ‘Ca. Liberibacter solanacearum’, a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

Download Full-text

Development of a real-time PCR assay for the detection and identification of Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri

Journal of Medical Microbiology ◽

10.1099/jmm.0.47235-0 ◽

2007 ◽

Vol 56 (10) ◽

pp. 1346-1349 ◽

Cited By ~ 17

Author(s):

Tadayuki Iwase ◽

Keiko Seki ◽

Hitomi Shinji ◽

Yoshimitsu Mizunoe ◽

Shogo Masuda

Keyword(s):

Real Time ◽

Real Time Pcr ◽

High Specificity ◽

Pcr Assay ◽

Coagulase Negative Staphylococci ◽

Staphylococcus Warneri ◽

Staphylococcus Haemolyticus ◽

Staphylococcus Capitis ◽

Detection And Identification ◽

Species Specific

Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3′ end of the primer (3′ mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (101–107 cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.

Download Full-text

The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus

FEMS Microbiology Ecology ◽

10.1016/j.femsec.2004.09.002 ◽

2005 ◽

Vol 51 (2) ◽

pp. 257-264 ◽

Cited By ~ 101

Author(s):

Simon D. Atkins ◽

Ian M. Clark ◽

Sonal Pande ◽

Penny R. Hirsch ◽

Brian R. Kerry

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Paecilomyces Lilacinus ◽

Specific Primers ◽

Species Specific

Download Full-text

SIMPLIFYING CELIAC DISEASE PREDISPOSING HLA-DQ ALLELES DETERMINATION BY THE REAL TIME PCR METHOD

Arquivos de Gastroenterologia ◽

10.1590/s0004-28032015000200013 ◽

2015 ◽

Vol 52 (2) ◽

pp. 143-146 ◽

Cited By ~ 6

Author(s):

Nicole SELLESKI ◽

Lucas Malta ALMEIDA ◽

Fernanda Coutinho de ALMEIDA ◽

Lenora GANDOLFI ◽

Riccardo PRATESI ◽

...

Keyword(s):

Celiac Disease ◽

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

Melting Curve ◽

Melting Curve Analysis ◽

Specific Primers ◽

The Real ◽

Hla Dq ◽

Pcr Method

Background Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Genetic susceptibility is associated with two sets of alleles, DQA1*05 - DQB1*02 and DQA1*03 - DQB1*03:02, which code for class II MHC DQ2 and DQ8 molecules, respectively. Approximately 90%-95% of celiac patients are HLA-DQ2 positive, and half of the remaining patients are HLA-DQ8 positive. In fact, during a celiac disease diagnostic workup, the absence of these specific DQA and DQB alleles has a near perfect negative predictive value. Objective Improve the detection of celiac disease predisposing alleles by combining the simplicity and sensitivity of real-time PCR (qPCR) and melting curve analysis with the specificity of sequence-specific primers (SSP). Methods Amplifications of sequence-specific primers for DQA1*05 (DQ2), DQB1*02 (DQ2), and DQA1*03 (DQ8) were performed by the real time PCR method to determine the presence of each allele in independent reactions. Primers for Human Growth Hormone were used as an internal control. A parallel PCR-SSP protocol was used as a reference method to validate our results. Results Both techniques yielded equal results. From a total of 329 samples the presence of HLA predisposing alleles was determined in 187 (56.8%). One hundred fourteen samples (61%) were positive for a single allele, 68 (36.3%) for two alleles, and only 5 (2.7%) for three alleles. Conclusion Results obtained by qPCR technique were highly reliable with no discordant results when compared with those obtained using PCR-SSP.

Download Full-text

Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds

Phytopathology ◽

10.1094/phyto-12-14-0364-r ◽

2015 ◽

Vol 105 (9) ◽

pp. 1245-1249 ◽

Cited By ~ 11

Author(s):

Sebastian Kiewnick ◽

Jürg E. Frey ◽

Andrea Braun-Kiewnick

Keyword(s):

Real Time ◽

Real Time Pcr ◽

The United States ◽

Locked Nucleic Acid ◽

Coi Gene ◽

Root Knot Nematode ◽

Detection And Identification ◽

Meloidogyne Enterolobii ◽

Meloidogyne Spp ◽

Pcr Assays

Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.

Download Full-text