Page 7 - Microorganism Species Analysis
P. 7

DNA Sequencer                                                                                                                                                                                                        DNA Sequencer
                                                                 Genome Information
            Microorganism Solutions                                                                                                                                                                                              Microorganism Solutions




                     Identification of Bacteria by Gene Analysis                                                                          Data
                     The identification of microorganisms by gene analysis has become the general method in recent years. The Japanese
                     Pharmacopoeia shows a method of sequence analysis of part (approx. 300 bases) of bacterial 16S ribosomal DNA (rDNA).   Gene Analysis of Changes in Microorganism Communities During Water Treatment
                     However, in practice it may not be possible to clearly distinguish between extremely closely related types using sequence
                                                                                                                                           The treatment of factory effluent has been attracting greater attention in recent years as one approach to corporate   Microorganism Species  Observation of
                     information for only about 300 bases, and multiple types of bacteria can be identified as identical. It is believed that
                                                                                                                                           environmental management. Shimadzu installed and operates a wastewater treatment system that uses aeration. The
                     sequence analysis of the entire 16S rDNA (approx. 1,500 bases) is required for accurate identification of bacteria by gene
                                                                                                                                           example below shows the evaluation of changes in bacteria flora detected before and after the treatment process.
                     analysis.
            Observation of
                     Sequence decoding with a general DNA sequencer is adequate for a restricted number of samples, such as colonies of
                     specific bacteria produced by separation and cultivation. However, the throughput of a general DNA sequencer is too low to           Effluent from part washing  Defoamer       Defoamer
          Microorganism Species
                     conduct large-scale gene analysis, such as for the currently topical metagenome analysis that identifies microorganisms in
                     entire bacteria colonies. The DeNOVA-5000HT, which allows low-cost, long-chain decoding of multiple samples, is ideal for                                                               Treated wastewater
                                                                                                                                                     Kitchen effluent
                                                                                                                                                                           Constant   Primary        Secondary
                                                                                                                                                                           volume   treatment        treatment
                                                                                                                                                                            pump      tank             tank
                                                                                                                                                           Washing
                        Features of Microorganism Community Analysis Systems Using                                                                        wastewater                                        Compressor
                                                                                                                                                            tank
                        the DeNOVA-5000HT High-Throughput BioMEMS DNA Sequencer                                                                                                                                                                        Microorganism Species  Identification of
                         The DeNOVA-5000HT can decode long-chain sequences of 800 bases or more with high statistical reliability (QV >
                         30 (99.9% reliability)). It covers the entire 16S rDNA (approx. 1,500 bases) with two sequence reactions and                                         Feed pump
            Identification of
                         decoding from both termini.                                                                                                                                                              Drain outlet
                                                                                                                                                                                      Compressed air
                         The DeNOVA-5000HT can continuously analyze 384 samples over 24 hours to decode more than 4 Mb per day.                                 Raw water
          Microorganism Species
                         The DeNOVA-5000HT has an especially high capacity for decoding long chains, which are not handled well by other                          tank                                           (     indicates special nozzles)
                         genome sequencer systems.
                                                                                                                                                                       Fig. 1 Schematic Diagram of Wastewater Treatment System
                         BioSpec-nano is a specialized spectrophotometer for routine laboratory needs for nucleic acid research. It can
                         analyze samples of 1 to 2 µL. To start the analysis, simply drop the sample on to the target and click a button.   Test and research flow
                         MultiNA uses reusable microchips to achieve automated, high-sensitivity analysis of nucleic acid samples, such as   1. Take constant-volume wastewater samples at each stage before, during, and after treatment and extract the DNA
                         PCR amplified fragments and restriction enzyme digestion fragments. It allows high-performance, automated             from microorganisms in wastewater.
                         analysis of up to 120 analyses at 80 sec minimum cycle time.                                                      2. Amplify the 16S rDNA gene by PCR. Create a 16S rDNA library by gene cloning.                             Specific Microorganisms  Detection of
                                                                                                                                           3. Conduct qualification and quantitation of the PCR amplification products using MultiNA or BioSpec-nano.
                                                                                                                                           4. Use a DNA sequencer to decode the entire base sequence of the 16S rDNA gene (approx. 1,500 bases).
            Detection of
                                                                                                                                           5. Conduct BLAST searches based on the sequence data and verify against the bacteria 16S rDNA gene sequence
                                                                                                                                               database to identify the bacteria type by sequence homology.
          Specific Microorganisms
                                                                                                                                           Table 1 shows the bacteria types detected or identified in wastewater before, during, and after treatment.
                                                           Decoded sequence information
                                                                                                 Database search                           Multiple types of bacteria were detected in the wastewater environment, including several known environmental
                                                                                                 (BLAST search)                            bacteria.
                                                                                                                                           Gene analysis can easily capture changes in bacteria communities, such as significant increases in species during
                                                                                                                                           water treatment by aeration.
                                                                                         Microorganism sequence database search                                                                                                                          Analysis of Microorganism-Derived
                                                                                         Sequence homology search                                         Table 1 Number of Bacteria Detected in Wastewater Before, During, and After Water Treatment   Components
                                                                                         Multiple alignment analysis
                                                                                                                                                                                                        Treatment
                                                                                         Creation of genealogical tree
          Components
                                                                                                                                                                     BLAST Top HIT                 Before  During  After
                          DeNOVA-5000HT                                                                                                                              Ideonella sp.                   6     8    3
                          High-Throughput BioMEMS DNA Sequencer                                                                                                      Sphingomonas sp.                6     5    4
                                                                                                                                                                     Rhodobacter sp.                 4     4    2
            Analysis of Microorganism-Derived
                                                                                                                                                                     Asticcacaulis sp.               2     5    6
                                                                                                                                                                     Sinorhizobium                   2     3    4
                                                                                                                                                                     Brevundimonas sp.               2     0    3
                                                                                                                                                                     Diaphorobacter nitroreducens    0     4    1
                                                                                                                                                                     Leadbetterella sp.              0     0    3
                                                                                                                                                                     Brevundimonas diminuta          0     0    2
                                                                                                                                                                     Uncultured Bacterium PSB-M-1    0     2    0                                      Other Applications
                                                                                                                                                                     Uncultured Bacterium A0640      0     2    0
                                                                                                                                                                     Uncultured Bacterium 054 E02 B DI P58  2  0  0
                                                                                                                                                                     Uncultured Bacterium LP B54     4     3    0
          Other Applications
                          BioSpec-nano                 MCE-202 MultiNA                                                                                               Uncultured Bacterium HP1B26     3     2    0
                          Life Science Spectrophotometer  Microchip Electrophoresis System   MultiNA Microchips and Reagent Kits                                     Uncultured Bacterium           17    18    21
                                                       for DNA/RNA Analysis                                                                                          Other species                   9     5    11
          6                                                                                                                                                                                                                                              7
   2   3   4   5   6   7   8   9   10   11   12