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

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.

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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.

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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.

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

Plant Disease ◽

10.1094/pdis-09-14-0959-re ◽

2015 ◽

Vol 99 (11) ◽

pp. 1584-1589 ◽

Cited By ~ 8

Author(s):

Wenbin Li ◽

Zonghe Yan ◽

Mark K. Nakhla ◽

Andrea M. Skantar

Keyword(s):

Real Time ◽

Internal Control ◽

Real Time Pcr ◽

The United States ◽

Grass Species ◽

Specific Primers ◽

Accurate Identification ◽

Detection And Identification ◽

Stem Gall ◽

Species Specific

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.

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A new Real Time PCR with species-specific primers from Plasmodium malariae/P. brasilianum mitochondrial cytochrome b gene

Parasitology International ◽

10.1016/j.parint.2020.102069 ◽

2020 ◽

Vol 76 ◽

pp. 102069

Author(s):

Emilly Henrique dos Santos ◽

Lidia Yamamoto ◽

Wilson Domingues ◽

Silvia Maria di Santi ◽

Kelly Aparecida Kanunfre ◽

...

Keyword(s):

Real Time ◽

Cytochrome B ◽

Real Time Pcr ◽

Plasmodium Malariae ◽

Cytochrome B Gene ◽

Mitochondrial Cytochrome ◽

Specific Primers ◽

Mitochondrial Cytochrome B ◽

Mitochondrial Cytochrome B Gene ◽

Species Specific

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Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii

Archives of Microbiology ◽

10.1007/s00203-012-0809-y ◽

2012 ◽

Vol 194 (9) ◽

pp. 749-757 ◽

Cited By ~ 11

Author(s):

Catarina Churro ◽

Paulo Pereira ◽

Vitor Vasconcelos ◽

Elisabete Valério

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Cell Number ◽

Planktothrix Agardhii ◽

Species Specific

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Identification of L. casei and L. rhamnosus in the dairy products using Real-Time PCR assay

South Asian Journal of Experimental Biology ◽

10.38150/sajeb.4(5).p222-225 ◽

2015 ◽

Vol 4 (5) ◽

pp. 222-225

Author(s):

K. G. Li ◽

G. P. Pogossian ◽

A. K. Moldagulova ◽

E. E. Bekenova ◽

A. Abdikadirova ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Dairy Products ◽

Production Control ◽

High Efficiency ◽

High Specificity ◽

Industrial Test ◽

Fermented Dairy Products ◽

Species Specific ◽

Fermented Dairy

Lactobacilli are essential and important biological objects used in food pro-duction and medicine. One of the sufficient problems is fast, reliable and highly specific identification of lactobacilli in the scientific research and cur-rent production control. We represent two species-specific real-time PCR in the present study to discriminate L. rhamnosus and L. casei basing on the unique peptidoglycan-hydrolase genes p40 and p75 respectively. PCR pri-mers and probes were designed to provide high specificity discrimination via high temperature of PCR annealing stage. High efficiency of the reactions is provided by the size of amplified DNA fragments minimization. Reliable re-producibility of the target sequences amplification and fluorescence detec-tion provide a basis for the future creation of industrial test-systems for op-erational control in the production of fermented dairy products.

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Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

Journal of Food Protection ◽

10.4315/0362-028x-68.6.1217 ◽

2005 ◽

Vol 68 (6) ◽

pp. 1217-1221 ◽

Cited By ~ 27

Author(s):

PAVEL KRCMAR ◽

EVA RENCOVA

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Single Species ◽

Absolute Quantification ◽

Quantitative Detection ◽

Vertebrate Species ◽

Target Species ◽

Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

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Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya Utilizing a Novel Species-specific Real-time PCR Assay

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0003469 ◽

2015 ◽

Vol 9 (1) ◽

pp. e0003469 ◽

Cited By ~ 16

Author(s):

Robin H. Miller ◽

Clifford O. Obuya ◽

Elizabeth W. Wanja ◽

Bernhards Ogutu ◽

John Waitumbi ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Novel Species ◽

Plasmodium Ovale ◽

Pcr Assay ◽

Western Kenya ◽

Plasmodium Ovale Curtisi ◽

Species Specific

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Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.02082-07 ◽

2008 ◽

Vol 74 (10) ◽

pp. 3306-3309 ◽

Cited By ~ 24

Author(s):

Kazuhiko Maeta ◽

Tomoya Ochi ◽

Keisuke Tokimoto ◽

Norihiro Shimomura ◽

Nitaro Maekawa ◽

...

Keyword(s):

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Specific Test ◽

Specific Identification ◽

Specific Fluorescence ◽

Species Specific ◽

Poisonous Mushrooms

ABSTRACT Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

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