scholarly journals A Multiplex-PCR Method for Strain Identification and Detailed Phylogenetic Analysis of AY-Group Phytoplasmas

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 299-305 ◽  
Author(s):  
Shigeyuki Kakizawa ◽  
Yoichi Kamagata
Keyword(s):  
Phylogenetic Analysis ◽  
Multiplex Pcr ◽  
16S Rdna ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Single Copy ◽  
Strain Identification ◽  
Specific Detection

Phytoplasmas are plant pathogenic bacteria that cause devastating losses in the yield of diverse crops worldwide. Specific detection and strain identification of phytoplasmas is important to prevent the spread of phytoplasma-induced diseases. Hence, methods to rapidly detect these organisms are important for pest control. Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects because they are highly sensitive, easily handled, and have a variety of analytical secondary applications. The phytoplasma 16S rDNA was widely used as a target of the PCR detection method; however, further target genes and more rapid methods have been required for more specific detection of phytoplasmas. Here, we developed a multiplex-PCR system to amplify several phytoplasma genes. We designed 36 primers, based on the genome sequence of ‘Candidatus Phytoplasma asteris’, to amplify 18 single-copy genes covering wide regions of the phytoplasma genome. Nine genes could be simultaneously amplified in a single PCR. This multiplex-PCR was applied to DNAs from 10 phytoplasma strains belonging to the AY-group, and different amplification patterns were obtained between strains, suggesting that this method would allow us to differentiate phytoplasmas at the strain level. Direct sequencing was also possible after the multiplex-PCR amplification by a modified sequencing method. Detailed phylogenetic analysis was performed using concatenated sequences, and evolutionary relationships among four Japanese isolates were revealed, where these strains could not be distinguished by their 16S rDNA. Thus, this multiplex-PCR system is useful for rapid strain identification and detailed phylogenetic analysis of phytoplasmas.

369 AMPLIFICATION OF THE SRY GENE FOR SEXING OF BUFFALO (BUBALUS BUBALIS) EMBRYOS USING NESTED MULTIPLEX PCR

2007 ◽  
Vol 19 (1) ◽  
pp. 300
Author(s):  
M. Zhang ◽  
Q. Fu ◽  
W. S. Qin ◽  
H. Y. Zheng ◽  
Y. Q. Lu ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Internal Control ◽  
Pcr Amplification ◽  
Single Copy ◽  
Bubalus Bubalis ◽  
Sex Identification ◽  
Sry Gene ◽  
Embryo Sex ◽  
Pcr Method ◽  
Male Specific

In mammals, the Y chromosome-linked SRY gene is responsible for male sex determination. Therefore, a logical approach for embryo sex identification is to amplify the male-specific single-copy SRY gene. The objective of this study was to design specific primers for amplification of buffalo SRY gene and develop a reliable PCR method for sex identification of buffalo embryos. Genomic DNA was extracted from swamp buffalo (Bubalus bubalis) peripheral blood. A pair of primers based on the sequence of Holstein bovine SRY gene (forward, 52-GTTTGCCTTATGGATTTATT-32; reverse, 52-TCTACTTTAGCCTATTTG-32) was used to amplify whole buffalo SRY gene. This amplified fragment was isolated and constructed into plasmids for sequencing. Two pairs of primers, S1/S2 (forward, 52-CCATGAACGCCTTCATTTTGTG-32; reverse, 52-ACGAGGTCGATATTTATAGC CC-32) and S3/S4 (forward, 52-AAGCAGCTGGGCTATGAGTGGAA-32; reverse, 52-ACGAGGTCGATATTTATAGCCC-32), were designed based on the SRY sequence above. Simultaneously, the G3PDH gene was amplified to serve as an internal control for both male and female embryos. A multiplex-nested-PCR system was optimized by varying the following parameters individually: concentration of Mg2+, dNTPs, primers, and different cycles number. Twenty-seven IVF morulae were identified with the optimal PCR procedure after biopsy. Accuracy of PCR amplification was verified by dot blotting. The sex of 24 embryos fertilized with Y-sperm separated by flow cytometry was also examined. Results indicated that the optimal procedure of Nested-Multiplex-PCR consisted of 1.5 mM Mg2+, 100 �M dNTPs, 0.5 �M SA3/SA4 primers, and 0.25 �M GA3/GA4 primers, and 35 cycles. Accuracy of identification was 100% for 27 IVF morulae; 14 were judged as males and 13 were females. The result of blotting confirmed that the accuracy of amplification was 100%. The proportion of males was 83.3% (20/24) in embryos fertilized with Y-sperm. This confirms that the PCR system targeting the SRY gene can be used for accurate sex identification of buffalo embryos. This study was supported by grants from the Foundation of Guangxi Key Laboratory for Subtropical Bio-Resource Conservation and Utilization (SB0403) and the Guangxi Department of Science and Technology (0626001-3-1).

Microbial 16S rDNA diversity in an anaerobic digester

1997 ◽  
Vol 36 (6-7) ◽  
pp. 49-55 ◽  
Author(s):  
Jean-Jacques Godon ◽  
Emmanuelle Zumstein ◽  
Patrick Dabert ◽  
Frédéric Habouzit ◽  
René Moletta
Keyword(s):  
Phylogenetic Analysis ◽  
Community Structure ◽  
16S Rdna ◽  
Bacterial Community Structure ◽  
Bacterial Population ◽  
Pcr Amplification ◽  
Operational Taxonomic Unit ◽  
Fluidized Bed Reactor ◽  
Rdna Sequences ◽  
Rdna Clone

The bacterial community structure of a fluidized bed reactor fed by vinasses was analysed by molecular identification. After PCR amplification, three 16S rDNA clone libraries of Bacteria, Archaea, and Procarya populations were established. Community structure was determined by phylogenetic analysis of 556 partial rDNA sequences (about 500 bp long). 139 OTUs (Operational Taxonomic Unit) were found among which 133 and 6 were from the Bacteria and Archaea domains respectively. The majority of bacterial OTUs are not closely related to all other hitherto-determined sequences. The ratio Archaea/Bacteria is 1/4 and the most frequent bacterial OTU represents less than 5% of the characterised bacterial population.

scholarly journals Identification of the most common pathogenic bacteria in patients with suspected sepsis by multiplex PCR

2014 ◽  
Vol 8 (04) ◽  
pp. 461-468 ◽  
Author(s):  
Mohammad Reza Arabestani ◽  
Hossein Fazzeli ◽  
Bahram Nasr Esfahani
Keyword(s):  
Multiplex Pcr ◽  
Internal Control ◽  
Predictive Value ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Clinical Samples ◽  
Coagulase Negative Staphylococci ◽  
Acinetobacter Baumanii ◽  
Suspected Sepsis ◽  
Microbiological Methods

Introduction: Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumanii have been found to be the most prevalent bacteremia-causing bacteria in patients with septicemia. Early detection of bloodstream infection (BSI) is crucial in the clinical setting. A multiplex PCR method for identification of these agents in clinical samples has been developed in parallel by conventional microbiological methods. Methodology: The target genes selected for each of the organisms were very specific for designing primers. Design of primers was done using Mega4, Allel ID6, Oligo6, and Oligo analyzer software. The test comprises a universal PCR from the 16S rDNA gene and multiplex PCR from the rpoB, gyrA, sss, and chromosome X (as an internal control). Results: The sensitivity and specificity for universal PCR and multiplex PCR in comparison with BC were 83.87% and 91.58%, and 74.19% and 91.58%, respectively. The positive predictive value (PPV) and the negative predictive value (NPV) for these two PCRs were 76.47% and 94.57%, and 74.19% and 91.58%, respectively. PCR failed to identify bacteria which were found conventionally in only 3.96% and 6.34% of the cases by universal and multiplex PCR (mostly bacteria not included in the PCR cassette). In 6.34% of the cases, multiplex PCR afforded identification of bacteria, but BC showed no bacteria in the sample. Conclusions: The multiplex PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Rapid detection of bacteria by multiplex PCR appears to be a valuable tool, allowing earlier pathogen-adopted antimicrobial therapy in critically ill patients.

Microfloral diversity of cultured and wild strains of Psoroptes ovis infesting sheep

Parasitology ◽  
2001 ◽  
Vol 123 (5) ◽  
pp. 441-446 ◽  
Author(s):  
J. C. HOGG ◽  
M. J. LEHANE
Keyword(s):  
Phylogenetic Analysis ◽  
Bacterial Diversity ◽  
16S Rdna ◽  
Pcr Amplification ◽  
Pantoea Agglomerans ◽  
Psoroptes Ovis ◽  
Sheep Scab ◽  
Pcr Product ◽  
Wild Strains

PCR amplification of 16S rDNA was used to determine the diversity of bacteria associated with 3 strains of sheep scab mite, Psoroptes ovis. Eight species of bacteria were identified by phylogenetic analysis of the PCR product sequences. Seven of these species are previously unreported in association with sheep scab mites. Five species were matched to Serratia marcesens, Proponibacteium acnes, Phyllobacterium rubiacearum, Pantoea agglomerans, Curacaobacter baltica, whereas the remaining 3 sequences matched unclassified sequences belonging to the gamma proteobacteria, pseudomonads and streptococci. Bacterial diversity of the in vivo cultured strain was very low and did not match the diversity of the 2 wild collected isolates. The diversity of the bacteria in relation to the disease of sheep scab and the possible importance of these bacteria in the diet of the mites are discussed.

Establishment of Multiple-PCR for Diagnosing Three Food-Borne Pathogenic Bacteria in Aquatic Products

2012 ◽  
Vol 554-556 ◽  
pp. 1029-1032
Author(s):  
Hui Li Xia ◽  
Hui Zheng ◽  
Jin Jun Zhang
Keyword(s):  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Pcr Amplification ◽  
National Standard ◽  
Aquatic Products ◽  
Gene Coding ◽  
Food Borne ◽  
Pcr Method

A rapid multiplex-PCR method was established in order to detect three foodborne pathogenic bacteria including Salmonella typli，Vibrio parahaemolyticus and Listeria monocytogenes in aquatic products. Three pairs of oligonucleotide primers were designed for multiplex-PCR amplification according to gene coding invasion protein A of Salmonella typli，gene coding immune response of Vibrio parahaemolyticus and regulation histone gene of Listeria monocytogenes. The amplified fragment sizes of these three bacteria were 213 bp, 369 bp and 517bp, respectively．The specificity of the multiplex- PCR was high and the minimum detection limit reached 103 CFU/mL. The multiplex-PCR method was used to analyze three aquatic product samples compared with national standard methods, the coincidence rate of two methods reached 100%. The method developed in this study had high sensitivity and specificity, which could be applied for the rapid detection and molecular epidemiology survey of food-borne pathogenic bacteria in aquatic products．

Rapid and Simultaneous Detection of Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli in Fish by Multiplex PCR

2020 ◽  
Vol 15 (2) ◽  
pp. 53
Author(s):  
Radestya Triwibowo ◽  
Novalia Rachmawati ◽  
Dwiyitno Dwiyitno
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Target Genes ◽  
Simultaneous Detection ◽  
Salmonella Spp ◽  
Fish Products ◽  
Confirmatory Assay

Pathogenic bacteria are commonly found as natural contaminants in seafood and fish products. Globally, several countries have been imposing strict regulations on the maximum levels of pathogens and consequently require microbial testing of pathogens before the products can be marketed. A culture-based method with biochemical assay has been widely used to detect pathogenic bacteria in food, despite its long and extensive process. Meanwhile, the alternative molecular-based method to overcome this problem, cannot differentiate between viable and nonviable cells, which may lead to underestimation. This study aimed to develop a multiplex PCR (mPCR) method as a confirmatory assay for the culture-based method to detect pathogens in fish products simultaneously. This method applied a pre-enrichment step to ensure the growth of low-level pathogens and the injured cells in the sample. The target genes were ToxR, InvA, and UidA for Vibrio parahaemolyticus, Salmonella spp. and Escherichia coli, respectively. This assay also amplified the 16S rDNA gene of bacteria as an internal control for the PCR reaction. By implementing liquid-based DNA extraction during analysis, the developed-mPCR was comparable to detect the targeted bacteria in artificially-contaminated samples. The method was more sensitive in naturally-contaminated samples, where the number of E. coli, Salmonella spp. and V. parahaemolyticus detected were 28, 7, and 22, respectively. While the conventional method only detected 26, 5, and 19 of the respective pathogens. With a relatively shorter time and lower operation cost, the mPCR method is potential as an alternative for the culture-based method.

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