Design of specific primer sets for SARS-CoV-2 variants using evolutionary algorithms

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

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Molecular Verification of Bloom-forming Aphanizomenon flos-aquae and Their Secondary Metabolites in the Nakdong River

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph15081739 ◽

2018 ◽

Vol 15 (8) ◽

pp. 1739 ◽

Cited By ~ 4

Author(s):

Hae-Kyung Park ◽

Mi-Ae Kwon ◽

Hae-Jin Lee ◽

Jonghee Oh ◽

Su-Heon Lee ◽

...

Keyword(s):

Secondary Metabolites ◽

Water Samples ◽

Its Region ◽

Cyanobacterial Blooms ◽

Specific Primer ◽

Nakdong River ◽

Off Flavors ◽

Primer Sets ◽

The Nakdong River ◽

Harmful Cyanobacterial Blooms

Aphanizomenon spp. have formed harmful cyanobacterial blooms in the Nakdong River during spring, autumn, and now in winter, and the expansion of blooming period and area, associated with the global warming is predicted. The genus Aphanizomenon has been described to produce harmful secondary metabolites such as off-flavors and cyanotoxins. Therefore, the production of harmful secondary metabolites from the Aphanizomenon blooms in the Nakdong River needs to be monitored to minimize the risk to both water quality and public health. Here, we sampled the cyanobacterial blooms in the Nakdong River and isolated ten Aphanizomenon strains, morphologically classified as Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888. Phylogenetic analysis using 16S rRNA and internal transcribed spacer (ITS) region nucleotide sequences confirmed this classification. We further verified the harmful secondary metabolites-producing potential of A. flos-aquae isolates and water samples containing cyanobacterial blooms using PCR with specific primer sets for genes involved in biosynthesis of off-flavor metabolites (geosmin) and toxins (microcystins, saxitoxins and cylindrospermopsins). It was confirmed that these metabolite biosynthesis genes were not identified in all isolates and water samples containing only Aphanizomenon spp. Thus, it is likely that there is a low potential for the production of off-flavor metabolites and cyanotoxins in Aphanizomenon blooms in the Nakdong River.

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Establishment of an Analytical System for the Human Fecal Microbiota, Based on Reverse Transcription-Quantitative PCR Targeting of Multicopy rRNA Molecules

Applied and Environmental Microbiology ◽

10.1128/aem.01843-08 ◽

2009 ◽

Vol 75 (7) ◽

pp. 1961-1969 ◽

Cited By ~ 160

Author(s):

Kazunori Matsuda ◽

Hirokazu Tsuji ◽

Takashi Asahara ◽

Kazumasa Matsumoto ◽

Toshihiko Takada ◽

...

Keyword(s):

Quantitative Pcr ◽

Intestinal Microbiota ◽

Reverse Transcription ◽

Rrna Gene ◽

Specific Primer ◽

Human Intestinal Microbiota ◽

Reverse Transcription Quantitative Pcr ◽

Primer Sets ◽

Analytical System ◽

Pcr Targeting

ABSTRACT An analytical system based on rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) was established for the precise evaluation of human intestinal microbiota. Group- and species-specific primer sets for Clostridium perfringens, Lactobacillus spp. (six subgroups and three species), Enterococcus spp., and Staphylococcus spp. targeting 16S rRNA gene sequences were newly developed for the quantitative analysis of such subdominant populations in human intestines. They were used together with previously reported group-specific primer sets for Enterobacteriaceae, Pseudomonas spp., and six predominant bacterial groups (the Clostridium coccoides group, the Clostridium leptum subgroup, the Bacteroides fragilis group, Bifidobacterium spp., the Atopobium cluster, and Prevotella spp.) for the examination of fecal samples from 40 healthy adults by RT-qPCR with lower detection limits of 102 to 104 cells per g of feces. The RT-qPCR method gave data equivalent to those yielded by qPCR for predominant populations of more than 108 cells per g of feces and could quantify bacterial populations that were not detectable (Staphylococcus and Pseudomonas) or those only detected at lower incidences (Prevotella, C. perfringens, Lactobacillus, and Enterococcus) by qPCR or the culture method. The RT-qPCR analysis of Lactobacillus spp. at the subgroup level revealed that a subject has a mean of 4.6 subgroups, with an average count of log10(6.3 ± 1.5) cells per g of feces. These results suggest that RT-qPCR is effective for the accurate enumeration of human intestinal microbiota, especially the entire analysis of both predominant and subdominant populations.

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Development of a quantification method with real-time PCR for three Pratylenchus species causing damage to chrysanthemum in Japan

Nematology ◽

10.1163/15685411-00002984 ◽

2016 ◽

Vol 18 (6) ◽

pp. 687-695 ◽

Cited By ~ 8

Author(s):

Yuki Koyama ◽

Koki Toyota ◽

Naoko Miyamaru ◽

Koichi Yoshida ◽

Kenta Uesugi

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Soil Samples ◽

Pratylenchus Penetrans ◽

Specific Primer ◽

Quantification Method ◽

Primer Sets

ThreePratylenchusspecies,P. penetrans,P. pseudocoffeaeandP. kumamotoensis, are major threats to chrysanthemum production in Japan. To develop a quantification method for these threePratylenchusspecies in soil using real-time PCR, we designed two new specific primer sets forP. pseudocoffeaeandP. kumamotoensisand applied the primer set NEG, developed previously, forP. penetrans. Relationships between the threshold cycle (Ct:y) values and number of nematodes inoculated (log2(no. (20 g soil)−1):x) were forP. penetrans,P. pseudocoffeaeandP. kumamotoensis. The quantification of thesePratylenchusspp. was conducted using 16 soil samples.Pratylenchus penetransandP. kumamotoensiswere detected from seven and one soil samples, respectively, while noP. pseudocoffeaewas detected. These results demonstrated that the presently designed primers are useful to quantify the densities of threePratylenchusspp. in chrysanthemum fields in Japan.

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Verification of complete bisulfite modification using Calponin-specific primer sets

Clinical Biochemistry ◽

10.1016/j.clinbiochem.2009.11.005 ◽

2010 ◽

Vol 43 (4-5) ◽

pp. 528-530 ◽

Cited By ~ 12

Author(s):

Ruethairat Sriraksa ◽

Patimaporn Chaopatchayakul ◽

Patcharee Jearanaikoon ◽

Chanvit Leelayuwat ◽

Temduang Limpaiboon

Keyword(s):

Specific Primer ◽

Primer Sets ◽

Bisulfite Modification

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Strategy for Modular Tagged High-Throughput Amplicon Sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.05146-11 ◽

2011 ◽

Vol 77 (17) ◽

pp. 6310-6312 ◽

Cited By ~ 16

Author(s):

Daniel Aguirre de Cárcer ◽

Stuart E. Denman ◽

Chris McSweeney ◽

Mark Morrison

Keyword(s):

High Throughput ◽

Amplicon Sequencing ◽

Modular Approach ◽

Specific Primer ◽

Bar Code ◽

Pcr Product ◽

Primer Sets

ABSTRACTThe use and validation of a strategy that allows a universal set of bar-coded sequencing primers to be appended to an amplified PCR product is described. The strategy allows a modular approach, in that the same bar code can be used with two or more target-specific primer sets, even simultaneously.

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Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira

Applied and Environmental Microbiology ◽

10.1128/aem.01775-20 ◽

2020 ◽

Vol 86 (20) ◽

Author(s):

Ran Jiang ◽

Jian-Gong Wang ◽

Ting Zhu ◽

Bin Zou ◽

Dan-Qi Wang ◽

...

Keyword(s):

Drinking Water ◽

Water Samples ◽

Water Systems ◽

Ammonia Oxidizers ◽

Specific Primer ◽

High Coverage ◽

Nitrite Oxidizing Bacteria ◽

Nitrite Oxidoreductase ◽

Primer Sets ◽

Drinking Water Systems

ABSTRACT Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems. IMPORTANCE Nitrospira is a dominant nitrite-oxidizing bacterium in many artificial and natural environments. The discovery of complete ammonia oxidizers in the genus Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A predominated over sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

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