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😷 | [8/23] New Corona Confirmed Infection of XNUMX New People


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[8/23] New Corona XNUMX newly confirmed infections

 
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In Nara City, 13 people have already been tested for PCR as concentrated contacts with this caregiver, and the remaining 10 have been confirmed negative.
 

The prefecture and Nara City announced today that eight new cases of coronavirus infection have been confirmed.Newly infected ... → Continue reading

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Polymerase chain reaction

Polymerase chain reaction(Polymerase Rensa Hanno,English: polymerase chain reaction) IsDNAA reaction or technique that amplifies a specific region of a sample by millions to billions of times. Take the initials in EnglishPCR method, Or simplyPCRIt is also called "Polymerase Chain Reaction" in English.

DNA polymeraseと 呼 ば れ る酵素Utilizing the function of, any cycle through a series of temperature changesgeneArea orgenomeCopy spaceExponentialTarget (Mouse arithmeticThe objective is to amplify from a small sample of DNA to a sufficient amount to study its details.[1][2][3].Medical,Molecular biology,Forensic medicineIt is a useful technology that is widely used in fields such asCarrie MarisInvented by (Kary Mullis)[4][5],Nobel PrizeWas awarded.

Since the PCR method was established,Sequencing, Experiments such as gene mutation induction become possible,Molecular genetics,Physiology,TaxonomyIn addition to being utilized in research fields such as, analysis of ancient DNA samples,Forensic medicine,Paternity testUsed inDNA typingIdentification of infectious agentsInfectionTechnology development related to diagnosis (Nucleic acid amplification test), etc. have advanced dramatically. Also, from the PCR methodReverse transcription polymerase chain reaction,Real-time PCR,DNA sequencingEtc. are derived and developed. Therefore, today the PCR methodBiology,Medical scienceIt is the basis of gene analysis in a wide range of fields including[6][7].

principle

The PCR method operates by a series of thermal cycles of raising and lowering the temperature of a DNA solution containing a mixture of reagents. In the repeated cycle of heating and cooling of this DNA sample, dissociation of double-stranded DNA, binding of primer,酵素Three reactions, DNA synthesis by reaction, proceed, and finally a large amount of DNA fragments in a specific region are replicated.

In the PCR method, in addition to the DNA sample to be amplified (template), a large amount ofPrimer(In the target DNA regionComplementaryShort single-stranded DNA with sequence (Oligonucleotide)) and free, a component of DNAnucleotideAndPolymeraseIs a type ofDNA synthase(DNA polymerase) ThreereagentTo use.

  1. The first step is to heat the double-stranded DNA of the DNA double helix at high temperature.DegenerationAnd physically separate into single-stranded DNA. The temperature at which denaturation occurs depends on the base composition and length (number of bases) of DNA, and generally longer DNA requires higher temperature.
  2. Next, the solution containing the single-stranded DNA is cooled, and the primer is bound to the complementary sequence site of the single-stranded DNA to partially form the double strand (annealing). When the cooling is rapid, it is difficult for long DNAs to recombine into double strands, but short DNA fragments (Oligonucleotide) Takes advantage of the fact that they can be easily combined. As a result, a primer is bound to a part of the target long single-stranded DNA. By keeping the primer overwhelmingly more than the DNA, the tendency of the DNA and the primer to bind becomes more predominant than the tendency of the DNA and the DNA to bind.
  3. Next, heat the solution slightly to remove this double-stranded DNA site.templateAsDNA polymeraseBy making the primer act as a starting point and releasenucleotideDNA that is complementary to the single-stranded part酵素Are synthesized. After the DNA is synthesized, the temperature is raised again to return to the first step, and this cycle is repeated from the first DNA denaturation to promote the amplification. As the PCR reaction progresses, the generated DNA itself is used as a template for replication, and the original DNA template isExponentialIs amplified toChain reactionAdvances.

As described above, the PCR method utilizes the difference in the rate of denaturation and annealing depending on the DNA chain length to repeat DNA synthesis simply by repeatedly raising and lowering the temperature of the reaction solution, and amplifying a partial region of any DNA. Is.

If the DNA polymerase used is heat-sensitive, the polymerase will denature along with the DNA at the high temperature of the denaturation step,DeactivationResulting in. Therefore, when the PCR method was first developed, DNA polymerase was added as an enzyme every time DNA was denatured, which was time-consuming and expensive.[8].. Currently,Thermus aquaticusTo sayThermophileIs a thermostable DNA polymerase derived fromTaq polymeraseBy using such as, it is possible to continuously proceed the reaction without adding an enzyme in the middle.

procedure

ready

The nucleotide sequences at both ends of the DNA region to be amplified are determined, and corresponding primers are artificially synthesized. At this time, the primer is a complementary sequence that binds to the 2'side of each strand of the double-stranded DNA to be amplified, and usually has about 3 bases. They are often custom made in the lab or can be purchased from a commercial biochemical supplier.

Reaction solution preparation

Amplification target DNA, primer, DNA polymerase and DNA synthesis material (Substrate)Deoxynucleotide triphosphate (dNTP), And a buffer solution to create an optimal salt concentration environment in which the enzyme works, and a PCR device (Thermal cycler). For the buffer solution included in the distributed PCR reagent kit,BivalentCationIs often included[9].NormallymagnesiumIon (Mg2+), but PCR-mediated DNA mutagenesis is highmanganeseIon (Mn2+) Can also be used to increase the error rate during DNA synthesis.[10].Taq In the case of DNA polymerase,As a monovalent cationpotassiumIon (K+) May be included[11].. In some casesAmmonium sulfateMay be added.Ammonium ion(NH4+) Has the effect of destabilizing the weak hydrogen bond between the mismatched primer and the template base pair, and can increase the specificity.[11].

PCR cycle

  1. 94 reaction solution° CHeat to about 30 seconds to 1 minute to keep double-stranded DNA into single strands (Fig. 2).
  2. 60° CRapidly cool to a certain degree (slightly different depending on the primer) andPrimerAnneal (Fig. ②).
  3. No separation of primersDNA polymeraseReheat to the optimum temperature range for the activity of. Depending on the experimental purpose, its temperature is 60-72° CIt is set to a degree. This temperature is usually maintained for 1 to 2 minutes depending on the length of time required for DNA synthesis and the length of amplification (Fig. XNUMX).
  4. Up to this point is one cycle, and by repeating the steps from ① to ③, a specific DNA fragment is amplified.

When the PCR treatment is repeated n times, 1 double-stranded DNAn-Amplify 2n times.However, since the maximum of 2 cycles is usually performed, the term of XNUMXn is approximately negligible.If the number of cycles is further increased, the DNA polymerase loses its activity over time and reagents such as dNTPs and primers are consumed, so that the reaction is limited and the series of reactions is finally stopped.

Points to remember

The success or failure of this reaction depends on the base sequences of the DNA to be amplified and the primer, each set temperature and time during the cycle, and the like. If they are inappropriate, they may amplify irrelevant DNA sequences or may show no amplification. In addition, there is a possibility that mutation will occur during the synthesis process, so it is necessary to check the nucleotide sequence of the product depending on the purpose of use.

Application of PCR

Amplification and quantification of DNA

PCR is a technique for dramatically amplifying a region of target DNA, and even a very small amount of DNA sample may be analyzed by passing through PCR. This proves that only trace amounts of DNA are availableForensic medicineIt is especially important in such fields.Or, for example, tens of thousands of years agoAncient DNAPCR is also effective for analysis of[12].

Quantitative PCR(Also called real-time PCR, or simply qPCR.RT-PCRNote that the amount of a specific DNA sequence present in a sample can also be estimated by[13].. this is,Gene expressionIt is used for purposes such as quantitatively determining levels. In quantitative PCR, the abundance of the target DNA region that originally existed is quantified by measuring the concentration of the PCR product that is amplified during the PCR cycle in real time during the PCR cycle process. can do. There are two major methods, one is non-specifically retained between the duplexes.fluorescenceAnother method is to use a dye, and the other method is to use a probe to which a fluorescent label is added in advance and which encodes a specific sequence. In the latter method, fluorescence can be detected only when the probe and its complementary DNA are hybridized.

Combined real-time PCR and reverse transcriptionRT-qPCR(Reverse transcription polymerase chain reaction), the method calledRNAIt is possible to quantify. In this technique, mRNA is the firstcDNAAnd the cDNA is quantified by qPCR. This method is often used to detect genes related to genetic diseases such as cancer and to measure expression levels.[14].

Application to biological research

PCRMolecular biology,GeneticsIt has been applied to various research fields including.

  • PCR can be used to selectively amplify and isolate specific DNA regions in the genome. Such use of PCR isSouthern blotting,Northern blottingIs widely used for the production of such hybridization probes and for DNA cloning that requires a large amount of DNA fragments derived from a specific DNA region.
  • PCRDNA sequenceIs often important in doing.. By various PCR, for example, a gene sequence or a DNA region to be analyzed can be extracted from a completely unknown genome and amplified.
  • PCRClassic experiments such as DNA cloningIt is often used in the process. For example, it is used when inserting a specific genomic region from a large genome into a vector. It can also be used to analyze or amplify DNA fragments already inserted in the vector. By partially changing the PCR protocol,mutationCan be artificially induced.
  • PCR is often used for research on ancient DNA. Most of such ancient DNA has been decomposed by ultraviolet rays and hydrolysis, and there is often only a trace amount of double-stranded DNA, so it is possible to analyze only by applying amplification by PCR. .. As an example of research that actually used PCR,NeanderthalsThe bone, 4 years oldMammothFrozen tissue ofEgypt OfmummyThere is a brain, etc.ロシアemperorAnd British KingRichard IIIIs being identified[12].. In some cases, it may be possible to restore the original DNA sequence to some extent by applying PCR amplification even to a DNA sample that has been considerably degraded.
  • Gene expressionPCR is also used to study the pattern. You can analyze body tissues and individual cells at various time-series stages to see which genes were activated/deactivated,Quantitative PCRCan also be used to quantify the actual expression level in detail.
  • PCRGenetic linkageIs also used for research. For example,Amplifies several loci from individual sperm simultaneously,MeiosisAfterChromosome crossoverStudies have been reported[15].. In this study, rare rare crossover events between very close loci were directly observed by analyzing thousands of sperm. Similarly, aberrant deletions, insertions, translocations, or inversions can be analyzed.
  • PCR can be used to induce site-directed mutagenesis of any gene or genomic region. By investigating these mutants, it is possible to elucidate, for example, the function of the protein, or to proceed with research for altering or improving the function of the protein.

Prenatal diagnosis

Whether a PCR is a carrier of a particular inheritance before the child is born, or actuallysickTest whether you are affected byPrenatal diagnosisCan be used for[16].Prenatal examinationDNA sample forAmniocentesisbyChorionic villus sampling, Or can be obtained by analysis of a very small amount of fetal cells circulating in the mother's bloodstream. PCR analysisPre-implantation diagnosisIs also essential and can test mutations in individual cells of the developing embryo.

Organ transplant tissue typing

PCROrgan transplantationEssential toOrganizational typingIt can also be used as a highly sensitive test.Blood TypeA proposal to replace the traditional antibody-based test with a PCR-based test against was also made in 2008[17].

Cancer genetic analysis

Many forms of cancer are associated with various types of cancer development.gene(Oncogene) ArrayWith variations, PCR technology could be used to analyze this mutation to tailor treatment strategies to patients. PCR alsoleukemia,LymphomaSuch asMalignantEnables early diagnosis of disease. It is being developed in the field of cancer research, and PCR is now routinely used. It has been reported that direct PCR assay of genomic DNA samples can detect translocation-specific malignant cells at least 10,000 times more sensitive than other methods[18].. PCR also allows the isolation and amplification of tumor suppressors. For example, it is possible to quantify single cells using quantitative PCR and analyze the abundance and combination of DNA, mRNA, and protein.[19].

Diagnosis of infectious disease

PCR aids in sensitive and rapid diagnosis of infectious diseases caused by bacteria and viruses[20].. In PCR,Mycobacteria ,Anaerobic bacteria, ま た はTissue cultureAssayAnimal modelfromウ イ ル スIt is also possible to quickly identify microorganisms that cannot be cultured or microorganisms that grow slowly. In addition, PCR diagnosis can distinguish between non-pathogenic strains and pathogenic strains by not only detecting infectious pathogens but also determining whether the bacterium has a specific gene[20][21].. On the other hand, various defects have been reported (Later).

  • Human immunodeficiency virus (HIV) Is a difficult target to discover and eradicate. The initial diagnosis of infection is the virus circulating in the bloodstream.antibodyHowever, the antibody does not appear until weeks after infection, the maternal antibody masks the infection of the newborn, and the amount of antibody does not change when treated with an HIV therapeutic agent. was there. Therefore, a high-sensitivity PCR method that can detect only one viral genome from over 50,000 cellular DNA samples was developed.[22].. This method enables early detection of infectious diseases, virus testing of donated blood, rapid infection testing of newborns, and quantification of antiviral treatment effects.
  • tuberculosis OfSome disease-causing microbes such as these are known to be difficult to sample from patients and grow slowly in the laboratory,cultureA lot of time and effort was spent in the base diagnosis. Tests by PCR can detect disease-causing microorganisms in samples, and from genetic analysisAntibiotic resistanceIt is possible to detect the presence or absence, etc., which may lead to the setting of an effective treatment policy and the evaluation of the treatment effect.
  • 家畜orwildThrough a group of animalsdiseaseBiology Diffusion and emergence of new toxic subtypes can be monitored by PCR testing.
  • By using a primer specific to the target sequence of viral DNA, viral DNA can be detected by PCR and also used for DNA sequencing. Due to the high sensitivity of PCR, virus detection may be possible immediately after infection and before the onset of disease[23].. Early detection may give the physician a significant lead time for treatment. The viral load contained within the patient can also be quantified by PCR-based DNA quantification techniques.
  • Social Services LizardIs caused by a bacterium called B. pertussis. This bacterium is characterized by a serious acute respiratory tract infection affecting a wide variety of animals and humans, leading to the death of many young children.Communicate and LizardBinds to cell receptors by two dimers and plays a role in cell immunityT lymphocyteIs a protein toxin that reacts with[24].. Since the sequence in the pertussis toxin gene can be detected by PCR, pertussis can be diagnosed very efficiently compared to the culture method.[25].

Application to forensics

PCR-basedDNA typing(Fingerprinting)Forensic medicineWidely applied in the field.

  • DNA typing is世界It is a technology that can uniquely distinguish one person from the entire population. Applying this technology, a small amount of DNA sampleCrime sceneIn some cases, suspects can be identified by collecting, analyzing, and comparing prisoners. A simpler method of use is to quickly exclude suspects during criminal investigations. Alternatively, test crime evidence decades ago to confirm the perpetration of convicted people, orImmunityIt also leads to doing.
  • Forensic DNA typing (forensic DNA typing) is an effective way to identify or exclude suspected crimes from the analysis of evidence found at crime scenes. The human genome has many repeat regions found within gene sequences or in noncoding regions of the genome. Specifically, up to 40% of human DNA is known to be repetitive[26].. There are two types of these repetitive non-coding regions in the genome, one called variable length tandem repeat (VNTR), which is 2-10 base pairs long, while the other is short tandem repeat (STR). It is called a repeat section of 100-2 base pairs. Then, PCR amplification can be performed using the primers flanking each repeat region. A statistically high probability of uniquely identifying each individual can be obtained by examining the size distribution of several STR fragments obtained from each individual.[26].. Furthermore, the complete sequence of the human genome has already been determined, and since this sequence information can be easily accessed from the NCBI website, various applications have been made. For example, the FBI has compiled a set of DNA marker sites used for identification, which are called the Combined DNA Index System (CODIS) DNA database.[26].. This database can be used to statistically determine the probability that a DNA sample will match. PCR is a very powerful and important analytical tool for forensic DNA typing because it requires very small amounts of target DNA for analysis. For example,Hair follicleHuman hair to which is attached contains enough DNA, if any, to perform the analysis. Similarly, severalspermA skin sample from under the fingernails, or a small amountbloodCan provide enough DNA for definitive analysis[26].
  • On the other hand, on the contrary, due to the format with low discriminationDNA fingerprintingIt is,Paternity testIs utilized for.. In this case, the subject, such as the body of an unidentified human, is compared to the DNA of the expected relatives, ie parents, siblings, children, etc. To identify the biological parents of the adopted (kidnapped) child,NewbornOf the actual biological father ofConfirm (Or excluded) is also used.
  • AmelogeninGene-based PCR enables real-time male and female sex determination from forensic bone samples. This allows you to determine the sex of ancient specimens and suspected criminals.[27].

PCR technical limitations

PCR has many advantages: the principle and the actual work are very simple, the results can be obtained quickly, and they are very sensitive.Quantitative PCR(qPCR, quantitative PCR) also has the advantage of being able to quantify the targeted DNA region.On the other hand, PCR is also known to have various technical restrictions and limitations.

One of the technical limitations of PCR is that it requires a priori knowledge of the sequence of the target region in order to generate primers that allow selective amplification.[28]..That is, the PCR practitioner usually needs to know the sequence information before and after the target DNA region in advance so that the primer and the template bind properly.Therefore, in principle, it is impossible to perform PCR on a target whose sequence information is completely unknown.Also, like any other enzyme, DNA polymerase itself is prone to errors during DNA synthesis and may mutate the sequence of the PCR amplification product produced.[29].. Furthermore, since PCR can amplify even a small amount of DNA, amplification may occur based on erroneously mixed DNA, resulting in ambiguous or erroneous results.

Many techniques and procedures have been developed to avoid these problems and optimize PCR conditions.[30][31].. For example, in order to minimize the possibility that the sample will be contaminated by the contamination of foreign DNA, by using separate rooms for each step of reagent preparation and PCR processing/analysis, they can be spatially separated. Separation is effective[32]..In addition, it is effective to always use disposable new tubes and pipette tips with filters to operate samples and reagents, thoroughly clean the workbench and equipment, and always work in a clean space.[33].. Reconsidering primer design and investigating the types of buffers and polymerase enzymes is also important in improving the yield of PCR products and avoiding the formation of spurious products. To the buffer systemFormamideAddition of reagents such as may increase PCR specificity and yield[34].. Computer simulation of theoretical PCR results (Electronic PCR) has also been developed to support primer design.[35].

Characteristics of PCR in diagnosis of infectious diseases

PCR is a very powerful and practical research tool, and indeed, in many infectious diseases, the etiology sequencing has been elucidated using PCR. This method is knownウ イ ル スIt also helps identify unknown viruses and contributes greatly to understanding the disease itself. If the procedure can be further simplified and a highly sensitive detection system can be developed, PCR will becomeClinical examinationIt is believed that it will take an important position in the room[36].. However, the use of PCR in the diagnosis of infectious diseases has been pointed out to have various disadvantages as well as advantages.

利 点

  • Human capital OfgenomeOnly specific DNA fragments (hundreds to thousands of base pairs) can be selectively amplified from a sample containing a long DNA molecule such as (30 billion base pairs).[1].
  • The purpose can be achieved with a very small amount of DNA.
  • Amplification reaction can be completed in a short time[2]..The time required for amplification is as short as 2 hours or less.
  • Testing is possible by using primers specific for each pathogen while keeping the equipment (PCR machine) used in common.[2].
  • Even if the pathogen is dead (even if it loses its infectivity), it can be amplified if the target nucleic acid is preserved, and even dangerous pathogens can be inactivated before testing.[2].
  • Sensitivity can be increased by using Nested PCR, real-time PCR, etc.[2].

Disadvantage

  • When biomaterials such as organs and tissues are used as samples, the detection rate differs depending on the sampling site and pretreatment of the material, and even if the result is negative, the presence of pathogens in the living body cannot always be denied.[2].
  • There is a limit to the amplification of nucleic acid due to the decrease in reaction efficiency due to generation of reaction inhibitors, etc., and the result will be negative if the nucleic acid cannot be amplified to a detectable amount. Cannot deny the existence of pathogens[2].
  • Testing of biomaterials such as blood and feces may be affected by the inhibitory substances in the test materials, so purification work is required, but it is not said to be complete.[2].. In addition, the storage condition of test materials, the number of freeze-thaw cycles, and the methods and conditions for nucleic acid purification can have a large impact on test efficiency and test results.[2].
  • Contamination from positive controls during testing, contamination from previous tests and experiments, and contamination of reagents with nucleic acids can lead to false positives (contamination)[2].

History and background

With Kjell KleppeH. Gobind KhoranaEt al. used an enzyme assay with primers and a short DNA template.vitroIn 1971Journal of Molecular BiologyFirst published in (Molecular Biology Journal)[37].. This was to explain the basic principle of PCR, but it did not receive much attention at that time, and the invention of the polymerase chain reaction was generally found in 1983.Mullis penaltiesIs considered to be due to[38].

When Mullis developed PCR in 1983, heCaliforniaEmeryvilleSo the firstBiotechnologyA company that is one of the companies (Cetus Corporation) Was working. Mullis said, "One night,Pacific coast highwayWhile driving in the car, I came up with the idea of ​​PCR."[39].. When he was thinking about a new method to analyze changes (mutations) in DNA, he tried to amplify a partial region of nucleic acid by repeating the DNA synthesis reaction using the already known oligonucleotide and DNA polymerase at that time. Came up with[39].

Mullis named this method "polymerase-catalyzed chain reaction",Nature,ScienceI submitted it as a paper to a famous scientific journal such as, but it was not published. On the other hand, the PCR method itself was made by a colleague of Cetus.Sickle cell diseaseIt was applied to a rapid diagnostic method for inherited diseases. It was reported as "Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia" in Science magazine, and it attracted the attention of scientists around the world before the original paper.[5].. Finally in 1987, Mullis's paper Methods in Enzymology Published as "Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction."[40].. Later MullisScientific Americanso,"PCR can start with a single molecule of genetic material DNA and produce 1,000 billion similar molecules in the afternoon. The reaction is easy to perform. It requires a test tube, some simple reagents, and a heat source. It is only"[41].. DNA fingerprinting in 1988Paternity testFirst used in[42].

In recognition of this achievement, Mullis worked with his colleagues at Cetus in 7, seven years after demonstrating PCR technology.Nobel Prize in ChemistryWon[43].. In addition, “Enzymatic Amplification of β-globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia” by RK Saiki and HA Erlich in 1985 (“Enzymatic amplification of β-globin genomic sequence for diagnosis of sickle cell anemia”). And restriction site analysis'')American Chemical SocietyReceived Chemistry Breakthrough Award in the History of Chemistry category[44][45].. However, some controversy remains regarding the contributions of other scientists to Mullis' work and whether he was the only inventor of the PCR principle, as described below.

PCR was initiallyE. coli5'-3' exonuclease activity was removed by subtilisin treatment of DNA polymerase IKlenow FragmentMost of them used to cause a reaction. However, this enzymecopyThis enzyme had to be added manually after each thermal cycle because it could not withstand the high temperatures required to separate post-cycling DNA double helices and deactivate the DNA polymerase.[46].. As a result, the initial steps of DNA replication were very inefficient and time consuming, requiring large amounts of DNA polymerase and continuous treatment throughout the process. The Cetus research group lives in a high temperature environment (hot spring) of 50 to 80°C to solve this drawback.Thermophilic bacteriumIsThermus aquaticus[47]As a thermostable DNA polymeraseTaq polymeraseWas purified and the PCR method using this was published in Science magazine in 1976.[6].T. aquaticusDNA polymerase isolated from ° C (194 ° F) Stable at high temperature, and maintains activity even after DNA denaturation[48]Eliminates the need to add new DNA polymerase after each cycle[49].. This has opened the way to the simplification and automation of PCR reactions, and has led to the development of a widely applicable method.

In this way, the Cetus Group (including initially Maris) played a large role in the application and development of the PCR method.

However, it was Carey Maris who first conceived this method and showed direction, so MarisNobel Prize in ChemistryWas awarded in 1993. PCR technologyMullis penalties PatentWas acquired and was assigned to work in 1983 when Mullis invented the technology.TaqThe polymerase enzyme is also patent protected.DupontThere were several well-known lawsuits related to this technology, including the unsuccessful lawsuits filed. Swiss pharmaceutical companyEf Hoffman La RochePurchased a patent right in 1992, but the patent right now expires[50].

PCR types and application methods

Conventional PCR
Normal PCR that repeats the reaction for 1 to 25 cycles with one set of primers[2].
Real-time polymerase chain reaction (Real-Time PCR,Real-time PCR
A method of drawing an amplification curve in real time by emitting light from a nucleic acid fragment and detecting it using a dedicated optical instrument.It is used when quantitative rather than qualitative is required.[2].
Reverse transcription polymerase chain reaction (RT-PCR,Reverse transcription polymerase chain reaction
A method in which RNA is converted to cDNA by reverse transcriptase and then PCR is performed.[2].
Nested polymerase chain reaction(Nested PCR)
A method in which the PCR product amplified by PCR is used as a template for the next reaction and another PCR is repeated using another primer pair.[2].
Multiplex polymerase chain reaction (Multiplex PCR,Multiplex PCR
A method to simultaneously perform PCR reactions for multiple target nucleic acids (DNA) in a single reaction tube[2].
Amplified fragment length polymorphism(AFLP)
A method in which genomic DNA containing target nucleic acid (DNA) is cleaved with a restriction enzyme, a short double-stranded DNA (adapter) is bound to the cleaved end, and PCR is performed using its complementary primer.[2].
Loop-Mediated Isothermal Amplification (LAMP method
A method based on a completely different principle from conventional PCR[2][51].

References

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  2. ^ a b c d e f g h i j k l m n o p q "Precautions when using PCR in disease diagnosis”. Agricultural Research Organization. 2020/3/12Browse.
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Related item

Nara

Nara(Training) isNaraLocated in the north ofCity.. In NaraPrefectural office locationAndCore cityIs specified in.

Nara periodIt is called the ancient city because the capital was located in. Also京都Was also called Nanto.

Geography

Nara periodToHeijokyoWas placedAncient capitalAndTenpyo cultureIs known as a flowering land.

At present, Nara City is a wide-area city that occupies the northern part of Nara Prefecture at the same time.Nara BasinHits the northern end of. Eastern part of the cityYamato PlateauThe highlands of 300m to 600m above sea level continue. The north of the city is ancientHeijozanIn the hilly area called (Narayama)KyotoIs in contact with. Narasaka, which crosses Mt. Heijo and leads to Yamashiro, has been one of the important transportation routes since ancient times.

The city area is wide in the east and west, with (1) eastern mountainous areas, and (2) many cultural assets.International Tourism and Culture CityIn the Middle East with a face asCity center, (3) Osaka OfSatellite city-Bed townWith multiple faces, the atmosphere of the region and the orientation of the inhabitants show a difference from the western part, which has been developed as a residential area with the character of.

Origin of the city name

In olden times, the present Nara city area was called Sohori.

The whole area of ​​the city is flat terrain.As if they were evenThe terrain isNaraThe theory that it was the origin of the city name of "is influential. For more on the etymology,Nara # etymologySee.

The current Kanji notation is "Nara", but in old documentsNara""Neraku""HeijoIs also written.

climate

Seto Inland Sea climateInland climateAlso has. Because the city is located in a basin, there are large differences in temperature between summer and winter and one day.

  • Temperature-up to 39.3 °C (1994(6)May 8), Minimum -7.8 ℃ (1977(52)May 2
  • Maximum precipitation-196.5 mm (2017(29)May 10
  • Maximum instantaneous wind speed-47.2 m (1979(54)May 9
  • Deepest snow-21 cm (1990(2)May 2
  • Maximum number of summer days-153 days (1994(6))
  • Maximum number of summer days-85 days (1994(6))
  • Most hot days-30 days (2010(22))
  • Maximum number of tropical nights-20 days (2017(29))
  • Maximum number of winter days-90 days (1984(Showa 59))
Nara City (Nara Local Meteorological Observatory) Climate
Month123456789101112Years
Highest temperature record ° C (° F)18.9
(66)
23.9
(75)
25.9
(78.6)
30.5
(86.9)
32.7
(90.9)
35.8
(96.4)
38.1
(100.6)
39.3
(102.7)
36.9
(98.4)
32.0
(89.6)
26.8
(80.2)
24.9
(76.8)
39.3
(102.7)
Average maximum temperature ° C (° F)8.7
(47.7)
9.6
(49.3)
13.4
(56.1)
19.8
(67.6)
24.1
(75.4)
27.2
(81)
30.8
(87.4)
32.6
(90.7)
28.2
(82.8)
22.2
(72)
16.5
(61.7)
11.4
(52.5)
20.4
(68.7)
Average daily temperature ° C (° F)3.9
(39)
4.4
(39.9)
7.6
(45.7)
13.4
(56.1)
18.0
(64.4)
21.9
(71.4)
25.8
(78.4)
26.9
(80.4)
22.9
(73.2)
16.6
(61.9)
11.1
(52)
6.2
(43.2)
14.9
(58.8)
Average minimum temperature ° C (° F)−0.2
(31.6)
−0.1
(31.8)
2.3
(36.1)
7.4
(45.3)
12.5
(54.5)
17.5
(63.5)
21.8
(71.2)
22.6
(72.7)
18.8
(65.8)
12.1
(53.8)
6.4
(43.5)
1.9
(35.4)
10.3
(50.5)
Minimum temperature record ° C (° F)−7
(19)
−7.8
(18)
−5
(23)
−2.4
(27.7)
1.4
(34.5)
7.3
(45.1)
12.2
(54)
12.8
(55)
7.7
(45.9)
2.3
(36.1)
−2.6
(27.3)
−6.6
(20.1)
−7.8
(18)
Precipitation amount mm (inch)49.6
(1.953)
63.3
(2.492)
103.2
(4.063)
97.7
(3.846)
143.5
(5.65)
188.8
(7.433)
165.1
(6.5)
111.8
(4.402)
163.3
(6.429)
111.1
(4.374)
74.1
(2.917)
47.3
(1.862)
1,316
(51.811)
Snowfall cm (inch)2
(0.8)
4
(1.6)
1
(0.4)
0
(0)
------0
(0)
1
(0.4)
8
(3.1)
Average days of precipitation7.97.912.310.611.312.911.88.611.59.88.37.7120.6
Average monthlyDaylight hours116.7115.5147.4180.3184.8143.5162.7205.4150.3154.5134.5127.31,823
Source: Japanese Meteorological Agency (Average value: 1981-2010, extreme temperature: 1953-present)[3][4]


History

※About the history of the city area before the enforcement of the municipal system,NaraSee also

To the west of Nara city areaUwanabe TumulusA huge burial mound of the 5th century was built,Saki shield line burial moundsIs formed. 『WanashoLooks likeYamatoSoegami-gunYamamurago, Yangjugo, Yashimago, Ookago, Kasugago andSoge DistrictIt was the land of Saki and Torigai.

The area around the current city appears on the stage of Japanese history,710To Kyo FujiwaraからHeijokyoIt was after moving to. After that, although there were several short-term capital transfers,NagaokakyoGo to784Until then, this place was the center of Japan.NagaokakyoAfter moving to Tokyo,Todaiji Temple,Yakushiji Temple,Kofuku-ji TempleBuddhist temple forces such as remained in this area and were called the South Capital.

Kofukuji Temple is still in Yamato even in the middle agesGuardianIt's a huge jobManorThe Buddhist temple forces with the Rather, the power of the Great Temple had a great influence in times of war, and as a result, it suffered several wars. Two Great Buddha burning cases (Nanto burningBattle of the Great Buddha of Todaiji Temple) Is a symbolic event. But,Muromachi PeriodからSengoku periodTo other countries and nearby areasYamato BushidanThe power of the Great Temple diminished as a result of effective control.

Edo PeriodToEdo Shogunate OfNara magistrateHas been installedHeavenAsTokugawa familyBecame a direct territory of.Edo Period OfTeramachiThe atmosphere of Naramachi (Naramachi). In addition, the southern part of the present city areaIse country OfTsuhanEnclave (around Furuichi TownTodo familyTerritory). Also in the northeastern part of Nara city areaYagyu ClanWas the territory of.

Pacific WarInside is the sameReligious cityIsKyoto CityBecause it was not subjected to a large-scale air raid with21st century OfReiwaMany cultural heritage remains today.

History

Change of city limits

Meiji 22 YearMeiji 29 YearMeiji 31 Year12th year of TaishoShowa 2 YearShowa 15 YearShowa 25 YearShowa 26 YearShowa 28 YearShowa 30 YearShowa 32 YearShowa 43 YearHeisei 3Heisei 17Now
Nara
Soegami-gunNaraNaraNaraNaraNaraNaraNaraNara
Nara Town
Sabo
Soge DistrictIkoma-gun
Toto Village
Soegami-gun
Daianji Temple
East city
Soge DistrictIkoma-gun
FushimiFushimi Town
Tomio VillageTomi Town
Soegami-gun
Obiyashi VillageObiha Town
Meiji village
Gotaya Village
Tatsuichi
Peace Village
Tahara
Oyanagi Village
Yagyu Village
Tozato Village
Sakawa Village
Tsukise VillageTsukigase Village
Yamabe District
Harigabetsusho VillageTsumura*Tsumura*
Tosuke Nomura
  • TsumuraWas renamed in 3 (until 3, the indication of "Tsubaki" was "Show", after 3, the bias was "Ne")

Administration

Mayor

Successive mayors

NameInauguration dateRetirement dateRemarks
1Yasumoto Hikosuke1898(31th year of Meiji)May 21898 (31th year of Meiji)May 4
2Kirishima Shoyo1898 (31th year of Meiji)May 41898 (31th year of Meiji)May 7
3Yoshimori Omori1898 (31th year of Meiji)May 91902(35th year of Meiji)May 1
4Rita Taita1902 (35th year of Meiji)May 41905(38th year of Meiji)May 3
5Matsui Motojun1905 (38th year of Meiji)May 41908(41th year of Meiji)May 2
6Genkichi Kimoto1908 (41th year of Meiji)May 41911(44th year of Meiji)May 10
7Hisashi Nishijo1911 (44th year of Meiji)May 111919(Taisho 8)May 5
8Fukutaro Sagawa1919 (Taisho 8th year)May 71925(Taisho 14)May 3
9Hirokichi Oguni1925 (Taisho 14th year)May 81929(4)May 8
10Morita Uzaburo1929 year (Showa 4 year)May 81933(8)May 8
11Ishihara Zensaburo1933 year (Showa 8 year)May 91937(12)May 9
12Sadataro Matsui1937 year (Showa 12 year)May 101939(14)May 10
13Kiyoshi Taki1939 year (Showa 14 year)May 101945(20)May 12Experienced assistant
14Kiyoshi Ishikawa1946(21)May 61946 year (Showa 21 year)May 11Experienced assistant
15Kataoka Yasutaro1947(22)May 41951(26)May 4Experienced assistant
16Masatsugu Takagura1951 year (Showa 26 year)May 41967(42)May 4
17Tadamasa Kagida1967 year (Showa 42 year)May 51980(55)May 9
18Hiroshi Kiyama1980 year (Showa 55 year)May 91984(59)May 9Earnings/assistant experience
19Eizo NishidaMarch 1984, 59 (Showa 9)1992(4) April 9Experienced assistant
20Yasunori Okawa1992 (Heisei 4)/9/282004(16) April 9Earnings/assistant experience
21Tadashi Kageda2004 (Heisei 16)/9/282005(17)May 7
22Akira Fujiwara2005 year (Heisei 17 year)May 72009(21)May 7
23Gen Nakagawa2009 (Heisei 21)/7/31Incumbent

City hall

1889 (22th year of Meiji) Municipal systemNara town was established by the enforcement, and the government office was located in Tojibayashi town.Yagyu ClanPlaced in the ruins of a mansion[8].Municipal systemEven after the enforcement, there was a government building in the same area until 1977 (Showa 52). Moved to the current Nihon-Oji Minami 2-chome building on February 11, 1[9].. In Nara City, the city hall is called "Nara City Hall".

Parliament

City council

  • 39 people

Nara Prefectural Assembly

  • Electoral district Yamabe-gun, Nara-shi
  • 11 people

House of Representatives

ConstituencyRepresentative nameParty nameNumber of winsRemarks
Nara Prefecture 1st Ward(Nara City, excluding the former Tsumura area,Ikoma CityShigeru KobayashiLDP2Constituency
Sumio MabuchiConstitutional Democratic Party6Proportional revival
Nara Prefecture 2st Ward(OldTsumuraNara city in the area)Sanae TakaichiLDP8Constituency

Sister cities/partner cities

Domestic

sister city
Other
  • Japanese flagNational Monzen Town Summit -A conference held by local governments, tourism associations, and commercial personnel who have Monzen towns that have developed around shrines and temples across the country to revitalize the region and promote town development.

foreign

Economy

Since Nara City is the prefectural capital, many national institutions, various financial institutions, and branches of large corporations are concentrated.Kintetsu Nara Station,Shin-Omiya StationAre concentrated around. For companies headquartered in NaraNanto BankAnd develop bus business in the prefectureNara KotsuThere is.In addition, we will develop related businesses such as railway networks, department stores, and real estate throughout Nara Prefecture.Kinki Nippon Railway(Kintetsu) andKintetsu GroupHas a great influence on the economy of Nara City and is a group companyNara KotsuIt also has a close relationship with the tourism industry.

The annual number of tourists reaches about 1400 million, but in recent years Nara tourism has included Kyoto city and school trips.OsakaThe number of rooms and the occupancy rate of accommodation facilities are low compared to other countries.

Large commercial facilitiesNara family(Kintetsu Department Store),Paradis,Mi NaraThere,Takanohara StationPrevious,Gakken Nara Tomigaoka StationIn frontAEON MALLIs open.Also,Yamatokoriyama CityThere is a shopping mall that is open on the border with Nara City.In the shopping district in the cityTouko Shopping Street, Mochiido Center Town,Konishi Sakura Street Shopping Streetand so on.Restaurants etc.Kintetsu Nara Station-JR Nara StationAround,Sanjo street, There are many stores around Shin-Omiya Station, and various stores are located in front of Gakuemmae and along the main road of Oshikuma.

Traditional local industries such as brush and ink still exist, but their economic scale is not large. In the south of the cityDaiwa House Industry,Sekisui Chemical Industry,ス ケ ー タ ー, Daiichi Kako and other large factories of house makers and plastic product companies are in operation.

The development of suburban residential areas had been underway since before the war,1950Or later,Gakuenmae StationIn the area west of the Heijo Palace ruins centered on Kintetsu Group, the development of residential land led by private companies has begun in earnest.1972At the northern end of the city Heijo hillsAtJapan Housing Corporation Was the first large-scale project in KansaiNew townIs Heijo/Soraku New TownThe move-in to has begun.After that, residential land development progressed all over the suburbs of the city, especiallyKintetsu Nara Line,Kintetsu Keihanna Line ,Kintetsu Kyoto LineAlong the line is Osaka CityBed townBecause it is, commuting to Osaka City (so-called)Nara citizen) Is many.

2010Held inHeijo 1300 year festivalTaking this opportunity, a continuous grade crossing project around JR Nara Station was carried out, and the scenery of the city was in 1988.Nara Silk Road ExpoIt has changed significantly since then.At the same time, cultural assets such as the restoration of the Daigokuden of the Heijo Palace site and the restoration of the cathedral at Yakushiji and Kofukuji were also carried out.Since then,2020 Tokyo Olympics,2025 Osaka / Kansai Expo, Scheduled for 2037Linear Chuo ShinkansenAccommodation facilities are being developed in various parts of the city in anticipation of an increase in tourism demand due to the opening of all lines.

Headquartered major companies

National agency

Region

population

Demography29201.svg
Nara City and National Population Distribution by Age (2005)Nara City's Age and Gender Population Distribution (2005)
Purple-Nara
Green-All over Japan
Blue-male
Red-female

Nara city (equivalent area) population changes
Ministry of Internal Affairs and CommunicationsStatistics Bureau CensusThan

Looking at the population change from the previous survey from the 22 census (preliminary figure), it was 2010 people, a decrease of 0.97%, and the increase/decrease rate is 366,528th among 39 municipalities in the prefecture. Nara city population

  • Data source Nara Prefectural Statistics DivisionPopulation as of October 10, each year according to the survey.
  • As of October 2007, 10: 1 people
  • Population growth rate (2002 → 2007): -1.6%
*The population in 2005 is the total of Nara City, Tsukigase Village, and Tsumura Village.

Prefecture agency

Education

University/Junior college

National
Prefectural
private

high school

Prefectural
City
private
Closed School/Integration: Only after Heisei

Secondary school

Junior high school

National
City
private

primary school

National
City
private

communication

Tel

Post

The above is the collection and delivery station. *The postal codes in Nara are as follows.

  • "630-80xx""630-81xx""630-82xx""630-83xx""630-84xx==Nara city center, Aoyama, Amagatsuji, Nishi no Kyo, temple, Furuichi, Obiha district, etc. In charge of collection and delivery of Nara Central Bureau.
  • "631-00xx""631-08xx=Saidaiji, Ayameike, Gakuenmae, Tomio, Tomigaoka, Takanohara area, etc. In charge of collection and delivery of Nara Nishi station.
  • "630-11xx”= Sukawa, Sakawa area, etc. In charge of collection and delivery of the Sugawa station.
  • "630-12xx== Yagyu area etc. In charge of collection and delivery of the Yagyu Bureau.
  • "630-21xx] = Mizuma, Tahara area, etc. In charge of collection and delivery at the Makoto Department.
  • "630-23xx== Tsukigase Village area, former Soegami District. In charge of collection and delivery at Hatano Bureau (Yamazoe Village).
  • "632-01xx== Former Yamabe-gun, Tsumura Village area, generally north of Meihan National Highway. Responsible for collection and delivery of the Shinjuku Bessho Bureau.
  • "632-02xx== Old Tsumura area, generally south of Meihan National Highway. In charge of collection and delivery at Someda Bureau (Uda City).

Transportation

By trainJR West JapanArea is small, and the commuting demand in western residential areas isKintetsuIs in charge.Many roads in the city are narrow compared to the traffic volume, and chronic congestion occurs on holidays. The only city in which 47 prefectures are locatedHighway national roadHowever, it connects to central Osaka via a general toll road (Daini Hanna Road).In addition, it passes through the city where route formulation has been difficult for many years.Kyonawa Expressway(General highway 24Bypass) construction2019Was started.

Train

According to the JTB timetable, the central station is JR Nara Station, but Kintetsu Nara Station is better.Nara ParkClose to major tourist destinations such asBoarding and alighting personnelThere are overwhelmingly more Kintetsu Nara stations.Both stations are about 900m apart in a straight line.

West Japan Railway (JR West)

In addition to the above,Kizu Station(KyotoKizugawa) Is the end pointNara LineAlso, all the trains that get into the same station go to Nara Station via the Kansai Main Line.

*In the regular timetable, there are no high-quality trains that require a separate fare in addition to tickets for limited express trains. Nara Prefecture is the only prefecture in which 46 JR prefectures have railway lines and "no JR limited express train (passenger train/timer train) runs".[Annotation 2]In addition, the timetable was revised on March 2006, 3.KasugaThe only prefecture in 46 prefectures that "no JR excellent train (same as above) runs"[Annotation 2]Became. Similarly, after the abolition of the express "Kasuga", on the JR line in NaraRailcarNo passenger trains (commuter trains) run by[Annotation 3].. (The above are all as of September 2015)

Kinki Nippon Railway

Kintetsu Limited ExpressConnects the city to Osaka, Kyoto, Kashihara, and Ise-Shima, especiallyTokaido ShinkansenLimited express trains bound for Kyoto, which are responsible for transportation with and to Kyoto, are operated frequently.

bus

道路

highway

Pass through Nara cityDaini Hanna Toll Road,Meihan National Highway, In businessKyonawa ExpresswayAre all highways, not national highways, and are the only cities in 47 prefectures in Nara.Highway national roadDoes not exist. AroundNishi-Meihan Expressway OfKoriyama ICTenri ICThere is.

General national road

Main prefectural road

General prefectural road

Main road

Private toll road

空港

Tourism

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Scenic spot

[10]

Shrine

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Cliff buddha

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Facility

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amusement facilities

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サ ッ カ ー
basketball
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hockey
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Related person

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art

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athlete

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footnote

[How to use footnotes]

注 釈

  1. ^ At this point, Kozenin Town, Higashi-nosaka Town, Kitamimon Town, Imazaike Town, Imajiji Town, Tegai Town, Higashibuenaga Town, Higashisasaboko Town, Nakaomimon Town, Oshiage Town, Kawakubo Town, Noborioji Town, Yurugi Town, Minami Handa East Town, Kita Handa Higashi Town, Kiji Temple Town, Fukuchi-in Town, Kondo Hall Town, Bishamon Town, Ushi Town, Shiba Tombuki Town, Nakain Town, Tsurufukuin Town, Suspicious Tsujiko Town, Nain Town, Tsukijinouchi Town, Kawakami Torushuki Town, Jurin-in Town, Kawanoue Town, Jurin-in Hata Town, Yakushido Town, Ki Tsuji Town, Sannobu Town, Kyoshu Town, Nakatsuji Town, Wakido Town, Takamimon Town, Yinyo Town, Shonanin Town, Motokoji Town, Nishishinya Town, Shiba Shinya Town, Shimogimon Town, Nakashinya Town, Aji Manji Town, Kita Bath Town, Baba Town, Kitamuro Town, Minami Kido Town, Minami Naka Town, Minami Bath Town, Kotaro Town, Minamibukuro Town, Minami Shinmachi, Narukawa Town, Kawado Town, Hanazono Town , Inoue Town, Kyosetsu district east side town, Kyosetsu district west side town, Yanagi town, Nishijirin town, Komeiin town, Mochiaiden town, Nishikito town, Ogawa town, Minamiichi town, Tojibayashi town, Imamimon town, Ikeno town, Motorinin town, Tarui-cho, Hashimoto-cho, Higashi-Kido-cho, Tsubakii-cho, Minami-Uoya-cho, Teramachi, Okukomori-cho, Kitakomu-cho, Takaten-cho, Hankoku-cho, Hayashikoji-cho, Kado-shuri, Kado-shuri-ya, Kamisanjo-cho, Shimosanjo Town, Honkomori Town, Imazutsuko Town, Aburaka District Town, Nishinosaka Town, Millionga Tsujiko Town, Takatenshi Town, Sakashiniya Town, Okushiba Town, Kitaichi Town, Ibubuike Town, Higashi Shinka Town, Nishi Shinka Town, Nishi Shinka No., Funabashi Town, Boyashiki Town, Shujukuin Town, Nabeya Town, Soybean Town, Minamihoren Town, Uchimurahara Town, Kitakoji Town, Minami Handanaka Town, Handa Yoko Town, Kita Handa Nishi Town, Soybean Yamatochi Town, Kita Handaka Town , Kitaoya Higashi-cho, Tamon-cho, Nishi-Bakunaga-cho, Kitabukuro-cho, Nishi-Sasaboko-cho, Kitakawabata-cho, Kitao-ya Nishi-cho, Goto-cho, Oshikoji-cho, Handa-outuki-cho, Nishimimon-cho, Konishi-cho, Higashimuminami-cho, Higashimukinaka-cho, Higashimukita-cho, There are Hashiba town and Nakasuji town.
  2. ^ a b ShinkansenExceptHiroshimaAlso applies to this.
  3. ^ OtherTokyo-Kanagawa-ShizuokaAlso corresponds to this. JR is all electrifiedAichi-Ishikawa-Shiga-OsakaThere is a railcar regular passenger train in
  4. ^ With the closing, Nara City will be the only "Non-cinema" in Japan in 2012.Prefectural office location"[12], In the same yearYamaguchi Is the prefectural capital ofYamaguchi CityBut the only movie theater "Scary of Yamaguchi" closed[13][14]

Source

  1. ^ Establishment of Nara City Flag February 52, 2 Notification No. 10
  2. ^ Municipal emblem No. 36 of May 5, 5
  3. ^ "Normal value (value for each year/month)”. Japan Meteorological Agency. 2013/5/19Browse.
  4. ^ "1st to 10th place in the history of observation (value throughout the year)”. Japan Meteorological Agency. 2018/12/5Browse.
  5. ^ "Illustration of Japanese municipalities" Shogakukan dictionary editorial department,Shogakukan, January 2007, 1, first edition, first print, page 10.ISBN 4095263113.
  6. ^ Establishment of Nara City Flag
  7. ^ Mr. Nishitani, Deputy Mayor of Nara City(Mainichi Shimbun Nara Edition, September 2018, 9)
  8. ^ Shinji Okubo/Nara City History Editorial Board (Edit) "History of Nara City, Shishi IV" Chapter XNUMX Establishment of Nara City Section XNUMX Movement to a modern city Nara City Retrieved September 2018, 9
  9. ^ History of Nara-Showa (1926-1989)- Nara City Retrieved September 2018, 9
  10. ^ List of National Historic Sites, Scenic Spots and Natural Monuments Nara City. Retrieved November 2014, 11.
  11. ^ [Museum of Oriental Folklore] Uhufu Washa PinkAsahi Shimbun, September 2012, 2
  12. ^ “There is no movie theater in Nara city... Circumstances of “Japan's only prefectural capital””. MSN Sankei News (Sankei Digital). (September 2011, 7). オ リ ジ ナ ルArchived as of October 2011, 8.. https://web.archive.org/web/20110809195647/http://sankei.jp.msn.com/life/news/110719/art11071913160006-n1.htm 
  13. ^ "Goodbye, the silver screen of the prefectural capital, "Yamaguchi Scala", closed on the 1st of next month" "Asahi Shimbun," morning edition, October 2012, 10, Yamaguchi-26 region, page 1
  14. ^ "Scary of Yamaguchi, half-century history, pushed by cinemacon, DVD rental store". MSN Sankei News (Sankei Digital). (September 2012, 10). オ リ ジ ナ ルArchived as of October 2012, 10.. https://web.archive.org/web/20121030094314/http://sankei.jp.msn.com/region/news/121026/ymc12102602060000-n1.htm 
  15. ^ Grand sumo wrestling January 2nd place Place of Makuuchi's highest winning wrestler Deokatsu Ryuseki victory celebration parade and awarding of Nara citizen honorary prize Nara City (February 2020, 2) Read February 19, 2020

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