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

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

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

Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.42.12.5636-5643.2004 ◽

2004 ◽

Vol 42 (12) ◽

pp. 5636-5643 ◽

Cited By ~ 215

Author(s):

M. Rougemont ◽

M. Van Saanen ◽

R. Sahli ◽

H. P. Hinrikson ◽

J. Bille ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

18S Rrna ◽

Plasmodium Species ◽

18S Rrna Gene ◽

Rrna Gene ◽

Pcr Assays ◽

Species Specific

Download Full-text

Quantification of Blautia wexlerae and Blautia luti in human faeces by real-time PCR using specific primers

Beneficial Microbes ◽

10.3920/bm2014.0133 ◽

2015 ◽

Vol 6 (4) ◽

pp. 583-590 ◽

Cited By ~ 7

Author(s):

M. Touyama ◽

J.S. Jin ◽

R. Kibe ◽

H. Hayashi ◽

Y. Benno

Keyword(s):

Standard Deviation ◽

Real Time ◽

Real Time Pcr ◽

Rrna Gene ◽

Average Standard Deviation ◽

Specific Primers ◽

Human Faeces ◽

Faecal Samples ◽

Wet Weight ◽

Dominant Bacteria

The Clostridium coccoides group, including the genus Blautia and other genera, is one of the predominant bacterial groups in the human intestine. We re-examined 266 human faecal clones and 58 isolates in the C. coccoides group isolated by Hayashi et al. (2002) in order to elucidate the detailed distribution of Blautia wexlerae and Blautia luti in human faeces. Subsequently, we designed a primer pair specific for B. wexlerae and B. luti based on the 16S ribosomal RNA (16S rRNA) gene sequence. The number of B. wexlerae and B. luti in faecal samples of 12 healthy Japanese subjects was examined by real-time PCR assay. The number of the C. coccoides group in the 12 faecal samples was also determined using C. coccoides group-specific primers. Re-examination of the human faecal clones and isolates revealed that B. wexlerae and B. luti accounted for 19.5% of the clones and 25.9% of the isolates. B. wexlerae and B. luti were detected in all faecal samples with 5.3±3.2×109 cells/g faeces (wet weight, average ± standard deviation) as assessed by real-time PCR. Furthermore, B. wexlerae and B. luti constituted 32.3±12.7% (average ± standard deviation) of the C. coccoides group (1.7±0.8×1010 cells/g faeces). This demonstrates that B. wexlerae and B. luti were presented in human faeces with a high frequency as the dominant bacteria.

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

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.

Download Full-text

Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces

Applied and Environmental Microbiology ◽

10.1128/aem.01906-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6749-6756 ◽

Cited By ~ 62

Author(s):

Yun-Wen Yang ◽

Mang-Kun Chen ◽

Bing-Ya Yang ◽

Xian-Jie Huang ◽

Xue-Rui Zhang ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Bacterial Species ◽

Chronic Inflammatory Bowel Disease ◽

Trinitrobenzene Sulfonic Acid ◽

Rrna Gene ◽

Specific Primers ◽

Content Type

ABSTRACTMouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers forFirmicutes,Actinobacteria,Bacteroidetes,Deferribacteres, “CandidatusSaccharibacteria,”Verrucomicrobia,Tenericutes, andProteobacteriawere designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

Download Full-text

Simultaneous detection of 37 Lactobacillus species using a real-time PCR assay based on whole-genome sequence analysis

10.21203/rs.2.20588/v1 ◽

2020 ◽

Author(s):

Eiseul Kim ◽

Seung-Min Yang ◽

Bora Lim ◽

Si Hong Park ◽

Bryna Rackerby ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Genomic Analysis ◽

23S Rrna ◽

Comparative Genomic ◽

Rrna Gene ◽

Gene Sequences ◽

Species Specific

Abstract BackgroundLactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. ResultsTo select species-specific sequences or genes, a total of 143 Lactobacillus complete genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the real-time polymerase chain reaction (PCR) with the standard curve were confirmed. The real-time PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This Real-time PCR method was designed to detect 37 Lactobacillus species in a single reaction. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. ConclusionsThe method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

Download Full-text

Simultaneous detection of 37 Lactobacillus species using a real-time PCR assay based on whole-genome sequence analysis

10.21203/rs.2.20588/v2 ◽

2020 ◽

Author(s):

Eiseul Kim ◽

Seung-Min Yang ◽

Bora Lim ◽

Si Hong Park ◽

Bryna Rackerby ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

23S Rrna ◽

Lactobacillus Species ◽

Comparative Genomic ◽

Rrna Gene ◽

Gene Sequences ◽

Species Specific

Abstract Background Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the simultaneous identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. Results To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S–23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S–23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the real-time polymerase chain reaction (PCR) with the standard curve were confirmed. The real-time PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This Real-time PCR method was designed to detect 37 Lactobacillus species in a single reaction. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. Conclusions The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

Download Full-text