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Meningococcal disease describes infections caused by the bacterium that effect to brain and medulla with the high death rate about 10%20%. There are four main species related to this disease: Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogiens and Haemophilus influenzae. In which, Neisseria meningitidis causes the most in children. Yearly, in the world, the people die owing to meningococcal disease about 400.000 500.000 people. In Vietnam, the period from 2001 2010, an average of 650 recorded cases of the disease each year, mainly in the northern provinces. In 2012, there were 125 cases and in early 2013, sporadic cases found in several provinces and cities throughout the country, the risk of transmission in the community is still high. According to the Ministry of Health, in the first 6 months of 2015, the country recorded 24 cases of meningitis caused by Neisseria meningitidis, an increase of 16 cases over the same period last year. One of the reasons for the limited success of the strategy of vaccine and antibiotic therapy is the diversity of the serotypes serosubtype and rapid genetic transformation of Neisseria meningitidis through recombination making these bacteria resist the vaccine and antibiotic treatment. In Vietnam, the meningococcal disease precaution vaccine is currently imported from abroad.
VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS MOLECULAR CHARACTERIZATION OF NEISSERIA MENINGITIDIS STRAIN CIRCULATING IN THE NORTH OF VIETNAM HANOI, MONTH YEAR ACKNOWLEDGEMENT Fistly, I would like to dedicate my sincere thanks to my supervisor, Associ Prof Dong Van Quyen Vice director of Institute Biotechnology and my co-supervisor Nguyen Huu Duc, Ph.D Head of Department of Animal of Faculty of Biotechnology for their intellectual support though out this study Without their supervision, guardian and invaluable advice, I would not able to complete my study on time My heartfelt gratitude is dedicated to personnel from the Department of Molecular Microbiology of Institute of Biotechnology for supplying me the experimental bacteria Especially, Ms Le Thu Trang who directly guide me in process I conduct thesis My appreciation is extended to my friends for their wonderful advices, helps and supports in these years Last but not least my warmest gratitude to my parent Mr Pham Trong Truong and Mdm Duong Thi Anh, my lovely old sister Pham Thuy Phuong and my little sister Pham Thi Bich My Their continuous support, care and love have built me to be a better person Thanks for being with me always Student Pham Thi Ngoc CONTENTS TABLE OF CONTENTS FIGURE OF CONTENTS LIST OF ACRONYMS CSF: cerebrospinal fluid N.meningitidis: Neisseria meningitidis PCR: Polymerase Chain Reaction CHAPTER I: INTRODUCTION Meningococcal disease describes infections caused by the bacterium that effect to brain and medulla with the high death rate about 10%-20% There are four main species related to this disease: Streptococcus pneumoniae, Neisseria meningitidis, Listeria monocytogiens and Haemophilus influenzae In which, Neisseria meningitidis causes the most in children Yearly, in the world, the people die owing to meningococcal disease about 400.000- 500.000 people In Vietnam, the period from 2001 - 2010, an average of 650 recorded cases of the disease each year, mainly in the northern provinces In 2012, there were 125 cases and in early 2013, sporadic cases found in several provinces and cities throughout the country, the risk of transmission in the community is still high According to the Ministry of Health, in the first months of 2015, the country recorded 24 cases of meningitis caused by Neisseria meningitidis, an increase of 16 cases over the same period last year One of the reasons for the limited success of the strategy of vaccine and antibiotic therapy is the diversity of the serotypes / serosubtype and rapid genetic transformation of Neisseria meningitidis through recombination making these bacteria resist the vaccine and antibiotic treatment In Vietnam, the meningococcal disease precaution vaccine is currently imported from abroad Therefore, the vaccine may not be well compatible with the antigenic capacity of strains circulating in the country resulting in a decrease of the vaccines effectiveness Therefore, an urgent issue now is to study the distribution, molecular epidemiology and characteristics, genetic variation and evolution of Neisseria meningitidis strains circulating in Vietnam to have appropriate strategies to prevent human from meningococcal disease effectively Based on this fact, we carried out this research: ''Molecular characterization of Neisseria meningitidis strain circulating in the north of Vietnam’’ to define characteristics of these strains Results of the research will provide more information for further studies and especially to select suitable strains for vaccine production to protect human from meningococcal disease caused by Neisseria meningitidis meningococcus in Vietnam In this project, we will investigate, evaluate epidemic meningitis situation due to Neisseria meningitidis in the northern of Viet Nam; analyse the genetic characteristic, original evolution, genetic transformation in nucleotide and amino acid level of Neisseria meningitidis strains circulating in the northern of Vietnam The results of the study play important role in selecting strains to produce suitable vaccine, oriented to the import and indigenous vaccine production and meningitis epidemic precautionary strategy caused by Neisseria meningitidis in Viet Nam CHAPTER II: LITERATURE REVIEW 2.1 Meningococcal disease Meningococcal disease can refer to any illness that is caused by the type of bacteria called Neisseria meningitidis Neisseria meningitidis, often referred to as meningococcus, is a gram negative bacterium that can cause meningitis and other forms ofmeningococcal disease such as meningococcemia, a life-threatening sepsis also known as meningococcus These illnesses are often severe and include infections of the lining of the brain and spinal cord (meningitis) and bloodstream infections (bacteremia or septicemia) Meningitis and meningococcemia are major causes of illness, death, and disability in both developed and under-developed countries There are approximately 2,600 cases of bacterial meningitis per year in the United States, and on average 333,000 cases in developing countries The case fatality rate ranges between 10 and 20 percent.[1] The incidence of endemic meningococcal disease during the last 13 years ranges from to per 100,000 in developed countries, and from 10 to 25 per 100,000 in developing countries During epidemics the incidence of meningococcal disease approaches 100 per 100,000 The disease's pathogenesis is not fully understood The pathogen colonises a large number of the general population harmlessly, but in some very small percentage of individuals it can invade the blood stream, and the entire body but notably limbs and brain, causing serious illness Over the past few years, experts have made an intensive effort to understand specific aspects of meningococcal biology and host interactions, however the development of improved treatments and effective vaccines is expected to depend on novel efforts by workers in many different fields While meningococcal disease is not as contagious as the common cold (which is spread through casual contact), it can be transmitted through saliva and occasionally through close, prolonged general contact with an infected person Meningococcal vaccines have sharply reduced the incidence of the disease in developed countries Meningococcus bacteria are spread through the exchange of respiratory and throat secretions like spit (e.g., by living in close quarters, kissing) Meningococcal disease can be treated with antibiotics, but quick medical attention is extremely important Keeping up to date with recommended vaccines is the best defense against meningococcal disease 2.1.1 History of discovery Meningococcal disease was described by Vieusseux in 1805 during an outbreak with 33 deaths in the vicinity of Geneva, Switzerland [2] The Italian pathologists Marchiafava and Celli first described intracellular oval micrococci in a sample of CSF The Italian pathologists Marchiafava and Celli (1884) first described intracellular oval micrococci in a sample of CSF.[2]However, Anton Weichselbaum in 1887 first identified bacterium causing meningococcal disease in the CSF of six of eight patients of bacterial meningitis and the bacterium was named Neisseria Neisseria meningitidis causes a disease spectrum ranging from occult sepsis with rapid recovery to fulminant disease Before the 1920s, meningococcal disease was fatal in up to 70 percent of cases [3] Serum therapy with serum from immunized horses, introduced at the beginning of this century by Jochmann in Germany and Flexner in the United States, could reduce mortality from nearly 100% to 30%.[4], [5] The discovery of sulfonamides and other antimicrobial agents led to a further decline in case fatality rates Despite treatment with appropriate antimicrobial agents and optimal medical care, the overall case fatality rates have remained relatively stable over the past 20 years, at to 12%, with a rate of up to 40 % among patients with meningococcal sepsis.[6] Eleven percent to 19% of survivors of meningococcal disease have sequelae, such as hearing loss, neurological disability, or loss of a limb.[7] 2.1.2 Distribution Figure 2.1: Global Distribution of Invasive Meningococcal Desease by Serogroup 2.2 Neisseria meningitidis research situation and meningococcal disease in the world and in Vietnam 2.2.1 Worldwide Currently, in the world there are many studies from general to delve into the study of genetic characteristics At least complete genomes of Neisseria meningitidis strains have been determined and bring in Genbank For example: the genome of strain MC58 (serogroup B), strain H44/76 and strain NMA510612 (serogroup A) Most recent is complete Genome sequence of Neisseria meningitidis serogroup A strain NMA510612, 10 Hình 2.3 :Khuẩn lạc Neisseria meningitidis thạch chocolate [14] Hình 2.4: Khuẩn lạc Neisseria meningitidis thạch máu [14] 2.3.3 Resistance Meningococcal weak resistance: only live in CSF specimens approximately 3-4 hours after the body out Immediately destroyed by ultraviolet rays, the solution Chloramines B 0.5 to 1% or alcohol 70° At temperatures 55 / 60°C for 30 minutes or in / 10-minute brain tissue is destroyed Meningococcal weak resistance to dry conditions and light, easily killed by common disinfectants 2.4 Molecular characteristics 2.4.1 Target genes characteristics discovered Neisseria meningitidis 15 Some of the genes include: ctrA, Pora, crgA and 16 SrRNA already widely used in basic PCR assays [15], [22], [41] More specific, the gene sacB and siaD used widely from genogroup for almost: A, B, C, W135 and Y [16], [17] However, we know that there is no method to distinguish molecules 12 serogroups of Neisseria meningitidis of which detected only by antisera The change in the gene encoding the capsule in the chromosome has created various CPS in serogroup Capsule includes genes encoding regions A, B, C, D and E: - Areas A and C between galE and tex gene in chromosome [18] The A gene - encoding synthetic polysaccharide Zone B is the opposite direction of areas A or the sweep of C [19], in the B gene - is: Lipa and LipB related transport and surface expression of the capsule Zone C consists of genes (ctrA, -B, -C and -D), necessary for the transport to - the membrane capsule [20] Zone D consists of a series of genes (rmlA, -B, -C and, Gale), unrelated to the expression capsule, but is responsible for synthesis LOS (lipo-oligosaccharide) - [21] Region E only 01 genes, tex: synthetic adjust CPS [22] A regional gene sequences for individual differences of each serogroup, and the B, C, D and E have high conservatism between serum group (A, B, C, Y, W135, X, Z; 29E) [23] PorA (lớp 1-OMP) Phân nhóm huyết (VR1;VR2;VR3) 16 Polysascharide capsule Phenotype (B:NT:NT/P1.4/NT) PCR phát Nm (CtrA;CrgA) Nhóm huyết thanh: - SiaD (B;C;Y;W135) - mynB (A) Dịch tễ học phân tử: MLST; PorA;fetA PorB (lớp OMP) Nhóm huyết Figure 2.5: Characteristics of genes coding for antigens Neisseria meningitides Two target genes specific for Neisseria meningitidis species (CtrA and sodC) That is the gene which transported to the cell surface capsule CtrA gene had high conservative and often used in the PCR reaction [24] it is in the genes encoding the capsule (Figure 2), But no less than 16% loss in a carrier CtrA asymptomatic [25], [26] The trial the target gene does not alter Cu, Zn and Ca salts, which are SodC genes, not on the capsule gene coding, testing SodC direct detection of Streptococcus shell (encapsuleated), but it is completely used for the detection of meningococcal bacteria in humans carry genes in which no CtrA, which is why the need SodC additional set of genes discovered N meningitides Pora gene: Neisseria meningitidis class 1- OMP (membrane protein) antigen serum distinguish subgroups (KN candidate vaccines) [27], [28] Determination of nucleotide sequence of the gene part in the change zone Pora (VRS), VR1 and VR2 17 2.4.2 Target genes characteristics discovered serogroup of Neisseria meningitidis Neisseria meningitidis divided into 12 serogroups based on chemical structure data and bond between saccharide units of polysaccharide capsule that it express bacterium surface Serogroups are main cause disease, including : A, B, C, Y and W135, their structure are poly- α1-6 link with N- acetylmannosamine 6- phosphate capsule[29] The epidemic outbreak due to Neisseria meningitidis serogroup X, we express capsule is poly- α1-4- connected with N-acetylglucosamine 1-phosphate [30] has been studied and announced [31], [32] Serogroup D is not sorted into serogroups of N Meningitidis Sia gene synthesis sialic acid [33], called Syn capsule synthesis [34], is used to genotype and serogroup B (synD), C (SynE), Y ( synF) and W135 (syn G) SacB gene is the target gene for serogroups A and xcbA gene encode capsule polymerase that is target gene for serogroup X [35] The target gene described in synthetic capsule structure for serogroups A, B, C, Y, W135 and X (Figure 1) Most suitable primers used in sequencing, to determine serogroup by multiplex-PCR, real time PCR The capsule expression gene was identified in capsule operon: 01 of which encode synthetic capsule (called syn or sia gene, depending upon the nomenclature system used) and 03 for operon encoding transport capsule to the cell surface protein (ctr) Ctr gene product similar transport ATP in ABC species and high conservative serogroups cause disease PCR assays for the target gene's ctr first gene operon transportation capsule has been developed to detect Neisseria meningitidis encapsule and non-encapsule (nongroupable) although adding 01 specific genes and sensitivity of PCR assays were sodC gene The difference in the coding genome of Neisseria meningitidis capsule synthesis, the serogroup is easy basis to determine the synthetic capsule specific genes and genotyped of Nm [36] Serogroups Capsule Target Other A (α1-6)-N-acetyl-D-glucosamine gene name sacB nomenclature Myn B C W135 1phosphate (α1-8)-N-acetyneuraminic acid SynD (α1-9)-N-acetyneuraminic acid SynE 6-D-Gal(α1-4)-N-acetyneuraminic SynG acid(α2-6) 18 siaDB siaDC siaDw X (α1-4)-N-acetyl-D-mannosamine- xcbB Y 1-phosphate 6-D-Gal(α1-4)-N-acetyneuraminic SynF siaDy acid(α2-6) Table 2.2: Type of serogroup capsule and the target gene for genotype of Neisseria meningitidis Figure 2.6: Capsule gene map (CPS) of Neisseria meningitidis (37), ctrABCD operon coding ATP-protein (frame grid) SynABC (gray), D / E / F / G (dots), sacABCD (horizontal stripes), xcbABC (slash), encoding serogroup- specific enzymes for synthesis capsule oatC (serogroup C) and oatWY (W135 and Y serogroups), combined with syn operons and encoding O-acetyltransferases LipA and liB coding protein Ctre and ctrF known as Lipa and LipB Expression of poly- N-acetyl-D-mannosamine-1phosphate capsule of serogroup A dependent sacABCD operon (formerly known as mynABCD [37] Expression polyN-acetyl-D-glucosamine-1phosphate capsule of serogroup X depends on xcbABC opreron 2.4.3 Known strains of Neisseria meningitidis 19 Up to now, At least complete genomes of Neisseria meningitidis strains have been determined which encode about 2,100 to 2,500 proteins The complete genome sequence (GenBank accession number AE002098) was obtained by the random shotgun sequencing strategy [38] Neisseria meningitidis strain MC58 has a genome size of 2,272,351 base pairs (bp) with an average G1C content of 51.5% The genome contains four ribosomal RNA (rRNA) operons (16S-23S-5S) and 59 tRNAs with specificity for all 20 amino acids The 2158 ORFs identified represent 83% of the genome, with an average size of 874 bp Biological roles were assigned to 1158 ORFs (53.7%) with similarity to proteins of known function according to the classification scheme adapted from Riley [39] Three hundred and forty-five (16.0%) predicted coding sequences matched gene products of unknown function from other species, and 532 (24.7%) had no database match There were three major islands of horizontal DNA transfer found Two encode proteins involved in pathogenicity The third island only codes for hypothetical proteins They also found more genes that undergo phase variation than any pathogen then known Phase variation is a mechanism that helps the pathogen to evade the immune system of the host The genome size of H44/76 is 2.18 Mb, and 2,480 open reading frames (ORFs) were annotated, compared to 2.27 Mb and 2,465 ORFs for MC58 Both strains have a GC content of 51.5% Comparative analysis showed that four genes are uniquely present in H44/76 and nine genes are only present in MC58 Of all ORFs in H44/76, 2,317 (93%) show more than 99% sequence identity Of the 18 least-similar genes (90 to 95% sequence identity), [hmbR, tbp1, and an iron (III) ABC transporter] are associated with iron acquisition from the host In the genome sequence of MC58, a 32-kb region (NMB1124 to NMB1159) is duplicated (NMB1162 to NMB1197), while this region occurs only once in H44/76 (verified by PCR and 454 read coverage analysis) Obviously, the erythromycin cassette that truncates the siaD gene in MC58 is not present in H44/76 The complete sequence of the NMA510612 genome consists of one circular chromosome with a size of 2,188,020 bp, and the average G C content is 51.5% The 20 chromosome is predicted to possess rRNA operons, 163 insertion elements (IS), 59 tRNAs, and 2,462 CDS The NMA510612 genome was demonstrated to be highly collinear with that of WUE2594 (GenBank accession no FR774048), a strain of the ST5 genotype isolated from Germany in 1991 (9) The NMA510612 genome is smaller than the WUE2594 genome due to lack of a 42-kb Mu-like prophage region On the other hand, it specifically harbors several genes, such as tetA and hmbR Previous studies have demonstrated the presence of tetracycline resistance determinants like tet (M) and tet (B) The distribution of the hemoglobin receptor gene hmbR was investigated and observed at a significantly higher frequency among disease isolates than among carriage isolates In addition, we found that polymorphic regions exist in genes encoding type IV pilus proteins and type I restriction enzymes These variations may be involved in a rearrangement of surface-exposed proteins in this ST7 clone and enable the bacteria to overcome the herd immunity generated by the presence of the ST5 strain in the population CHAPTER III MATERIALS AND METHODS 3.1 Materials - Neisseria meningitidis strains for research serves selected from National Hospital of Tropical Deseases belong to Bach Mai Hospital -Primers ( em thưa Thầy,vì em chưa làm đến phần nên chưa biết trình tự mồi ạ) 3.2 Equipment and chemicals 3.3.1 Equipment Microwave, thermal cycler, horizontal electrophoresis system, analytical balance, voxter mixer, autoclave: - 80, and – 20 Benchtop freezer, biosafe cabinet, thermostatic water baths, pH meter, high speed centrifuge, eppendprf centrifuge Pippet, Eppendorf, tips,… 3.3.2 Chemicals 3.4 Methods 3.4.1 Total DNA extraction 21 DNA extraction is a process of purification of DNA from sample using a combination of physical and chemical methods The first isolation of DNA was done in 1869 by Friedrich Miescher Currently, it is a routine procedure in molecular biology or forensic analyses The procedure is suitable for all types of tissues from wide variety of animal, blood and plant species All DNA extraction steps are performed at weak acid pH (HEPES free acid) and optionally with hot chloroform for 'difficult' samples, and at room temperature The following protocol is designed for small and large tissue samples (tissue volume 100-200 μl) Protocol Step Grow colonies overnight at 37°C in shake cabinet with 5ml Luria- Bertani fluid medium Step Pour 1,5ml bacterium fluid overnight above into eppendorf 1,5ml Step Centrifuge at 10,000 rpm and 4ᵒC for Step cellular biomass collected by pouring off supernatant Step Add 400µl Lysis buffer Step Add K protein that finally concentration of K protein is 20ng/ml Incubate at 55 about hours in a recirculating water bath Vortex the well mixture briefly before incubate Step Add 400µl phenol: clorophorm: isoamyl (25:24:1) Vortex the well about 10 seconds Để yên 10 minitues Step Centrifuge at 12000 rpm and for 20 minutes Step Add 400µl isopropanol aim to precipitate DNA Then, incubate for about hour in a recirculating water bath Step10 Centrifuge at 12000 rpm and for 20 minutes to take DNA precipate Step11 Pour off supernatant, washing by alchol 70 After centrifuge 1000rpm and for 10 minutes - Dry tube contain DNA by in box or machine Step 12 Solution DNA in TE or H20 RNAse normally take about 20µl Step 13 The well mix and incubate at 37 about 30 half hour in a recirculating water bath 22 Step 14 agarose electrophoresis to test result 3.4.2 Primer design ( Em thưa Thầy, phần ạ, em chờ đến bước này, Thầy Quyền thiết kế mồi Sau đó, em hỏi xem Thầy Quyền dùng phương pháp, phần mềm ạ) 3.4.3 PCR amplification PCR is a molecular biology technique used to amplify one or a few copies of a piece of DNA exponentially, generating thousands to millions of copies of a particular DNA sequence Originally developed in 1983 by Kary Mullis, PCR is a technique popular and indispensable in the research laboratory medicine and biology with many different applications such as cloning DNA sequencing, researchers too Generating categories based on DNA evidence, or analysis of gene function; diagnosis of genetic diseases; identification of DNA fingerprints (used in forensic science and determine blood relations); detection and diagnosis of infectious diseases In 1993, Mullis was awarded the Nobel Prize for Chemistry along with Michael Smith for his research on PCR PCR-based thermal cycles, including the cycle rises and falls in reaction temperature DNA denaturation and DNA reproduction Primers (short DNA fragments are) bringing the complementary sequence to the target DNA sequence and the DNA polymerase is the key component that allows selectively amplify and repeat When the PCR process takes place, the new DNA is generated from the original DNA template will continue to be used as a template to synthesize DNA molecules next, forming a chain reaction sequence in which the DNA is amplified under exponentially • Overview of PCR 23 Figure 2.7: The principle of PCR Uses different temperatures to amplify DNA Step 1: Separate existing DNA strands – 95ºC (Denaturation) Step 2: Lower temperature to allow primers to bind to target DNA – 55ºC (Annealing) Step 3: Raise temperature to allow Taq Polymerase to build DNA strand – 72ºC (Extension ) The necessary tools and machinery used Sterile tube Stone pots - Pipetteman kinds (10μl, 20μl, 200μl, 1000μl) - First taper types (10μl, 20μl, 200μl, 1000μl) - Machine VOLTEX Cameras electrophoresis using UV Eppendorf PCR machine Vapo.protect The method of conducting: The ingredients and quantities in a tube: H2O: 19.1 µl Buffer: 2.5µl dNTPs: 0.2µl Primer F: 1µl Primer R: 1µl Taq : 0.2µl 24 DNA: 1µl Conducted 12 reactions, take the ingredients in the order in, mix the ingredients together and divide them into the tubes, each tube of 23.5µl Note: In the process of paying attention to the following points: + Always place the reaction tubes in ice + Centrifuge lightly the PCR mix after thawing and prior to the DNA sample + After the DNA into the mix, capping the tube, flick to the bottom tube to stir the mixture, then lightly centrifuged the tube before moving to the PCR machine + Restrict gas bubbling will cause errors in temperature 3.4.4 DNA Electrophoresis DNA on agarose gel electrophoresis technique was used to check the quality and quantity of DNA The principle of electrophoresis based on the characteristic structure of the DNA molecule Macromolecules that is negatively charged uniformly across the surface so influenced by an electric field intensity voltage and appropriate, we will move to the anode of the electric field The DNA segments will move in the electric field in the gel at a rate inversely correlated with their size In the same period of time, the baby will move DNA segments beyond the large segment, from which DNA will be separated into lines on gel electrophoresis When in electric field due to negatively charged molecules of DNA move towards the anode with different movement speed depends on the molecular weight The larger the DNA fragments to move more slowly And the protein molecules by different surface charge if the electrophoresis gel does not cause denaturation of the move in different directions with different speeds depending on the volume and surface charge If the gel electrophoresis in SDS after treatment with the protein surface due to become negatively charged, they move uniformly on the cathode of an electric field 25 Figure 2.8: The principle of electrophoresis Principle With agarose phase and immersed in 1X TBE buffer, PCR products are doublestranded DNA by bringing a lot of negative charges will be moving towards the anode and the movement will start at distance or near depending depending on the size of the PCR product is long or short After electrophoresis, the DNA of identical size will be concentrated into the bar, known as DNA bar, visible thanks to the double-stranded DNA was stained with ethidium bromide fluorescence the bar under ultraviolet light of a box UV lamps Based on the DNA ladder bar size that the size of the PCR products can be calculated or estimated Equipment and tools: - The DNA electrophoresis: power (80x120), shelf and electrophoresis tanks, wells produce electrophoresis comb, trays balance the tables by gel droplets - Electronic scales - Agarose cylinders to heat - Microwave - 2ml Eppendorf tube type - Sealed plastic tray size (10 20 30) to sell dyed gel - Shaker 26 Chemical composition + TBE 10X : Tris HCl, boric acid, EDTA Na2 + Agarose + Loading buffe: Bromo Phenol Blue, gycerol, Tris-EDTA + Ethidium bromide + DNA ladder: Thang ADN from 100 to 1000bps The method of conducing + TBE 1X solution phase by TBE 10X 100ml bottle contains built into a 900ml of distilled water, and mix Mix 30ml + 1% agarose agar by weight 0,3g 1X TBE agarose mixed in 30ml freshly brewed Melt the jelly in the microwave or double boiler until melted completely piglet, while occasionally shaking modular solution to ensure melt agarose Do not let the liquid boil agarose out + Prepare pour plaster molds, place comb into the mold, then pour plaster into the mold to achieve the desired thickness (about 4-5mm) To jelly in cold and room temperature characteristics + Place the jelly into the electrophoresis tray, pour 1X TBE solution for flooding the agar, remove the brush - For PCR tubes containing the "Deca PCR mix" and "NKYHV1 / GAV multiplex PCR mix" program has finished rotating heat, each tube received 7µl load buffering (loading buffer) Mix well - Download the PCR products were mixed with loading buffer to the wells in the agarose gel was immersed in 1X TBE buffer in electrophoresis tray Each load is about 15-20µl wells Always have a well for the DNA ladder - Electric cell with stable voltage of 125 volts for about 30 minutes of time or until the yellow line running on pole [+] starting at about 4cm away Read the results on the 312nm UV light box - Bring stained with ethidium bromide for 10 minutes toxic chemicals should be careful when handling, wear gloves and avoid inhaling the nose, and avoid skin contact, should the inside hood), 27 - Photograph gel electrophoresis and analysis of results 3.4.5 DNA sequencing DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA, The practical aspects revolve around designing and optimizing sequencing projects (known as "strategic genomics"), predicting project performance, troubleshooting experimental results, characterizing factors such as sequence bias and the effects of software processing algorithms, and comparing various sequencing methods to one another In this sense, it could be considered a branch of systems engineering or operations research The permanent archive of work is primarily mathematical, although numerical calculations are often conducted for particular problems too Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases [40] Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning This method's use of radioactive labelling and its technical complexity discouraged extensive use after refinements in the Sanger methods had been made Maxam-Gilbert sequencing requires radioactive labelling at one 5' end of the DNA and purification of the DNA fragment to be sequenced Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A + G, C, C + T) The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred 28 3.4.6 Phylogenetic tree analysis A phylogenetic tree or evolutionary tree is a branching diagram or "tree" showing the inferred evolutionary relationships among various biological species or other entities—their phylogeny based upon similarities and differences in their physical or genetic characteristics The taxa joined together in the tree are implied to have descended from a common ancestor Phylogenetic trees are central to the field of phylogenetic In a rooted phylogenetic tree, each node with descendants represents the inferred most recent common ancestor of the descendants, and the edge lengths in some trees may be interpreted as time estimates Each node is called a taxonomic unit Internal nodes are generally called hypothetical taxonomic units, as they cannot be directly observed Trees are useful in fields of biology bioinformatics, systematics, and phylogenetic comparative methods 29 such as [...]... electrophoresis and analysis of results 3.4.5 DNA sequencing DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine in a strand of DNA, The practical aspects revolve around designing and optimizing sequencing projects (known as "strategic... Note: In the process of paying attention to the following points: + Always place the reaction tubes in ice + Centrifuge lightly the PCR mix after thawing and prior to the DNA sample + After the DNA into the mix, capping the tube, flick to the bottom tube to stir the mixture, then lightly centrifuged the tube before moving to the PCR machine + Restrict gas bubbling will cause errors in temperature 3.4.4... serogroup A meningitis from the participating countries.[13] 11 2.2.2 In Vietnam The literature on the epidemiological situation meningococcal meningitis in Vietnam is still very limited According to some documents originally recorded epidemic meningococcal meningitis owing to N meningitidis belong to serogroup C cause in the southern provinces of Vietnam from 1977 to 1979 The death rate from an estimated... - Machine VOLTEX Cameras electrophoresis using UV Eppendorf PCR machine Vapo.protect The method of conducting: The ingredients and quantities in a tube: H2O: 19.1 µl Buffer: 2.5µl dNTPs: 0.2µl Primer F: 1µl Primer R: 1µl Taq : 0.2µl 24 DNA: 1µl Conducted 12 reactions, take the ingredients in the order in, mix the ingredients together and divide them into the tubes, each tube of 23.5µl Note: In the process... that there is no method to distinguish molecules 12 serogroups of Neisseria meningitidis of which detected only by antisera The change in the gene encoding the capsule in the chromosome has created various CPS in serogroup Capsule includes genes encoding regions A, B, C, D and E: - Areas A and C between galE and tex gene in chromosome [18] The A gene - encoding synthetic polysaccharide Zone B is the. .. menigigtidis in China that published in 2014 Beside research, some developmental nations produced vaccine to prevent For instance, In United States, a number of vaccines are available in the U.S to prevent meningococcal disease Some of the vaccines cover serogroup B, while others cover A, C, W, and Y.[8] A meningococcal polysaccharide vaccine (MPSV4) has been available since the 1970s and is the only meningococcal... meningitidis serogroups C and Y, and Haemophilus influenzaetype b (Hib) It was the first meningococcal vaccine that could be given to infants as young as six weeks old.[10] In October 2014 the FDA approved the first vaccine effective against serogroup B, named Trumenba, for use in 10- to 25-year-old individuals.[11] In 2010, the Meningitis Vaccine Project introduced a vaccine called MenAfriVac in the. .. detect Neisseria meningitidis encapsule and 1 non-encapsule (nongroupable) although adding 01 specific genes and sensitivity of PCR assays were sodC gene The difference in the coding genome of Neisseria meningitidis capsule synthesis, the serogroup is easy basis to determine the synthetic capsule specific genes and genotyped of Nm [36] Serogroups Capsule Target Other A (α1-6)-N-acetyl-D-glucosamine gene... to check the quality and quantity of DNA The principle of electrophoresis based on the characteristic structure of the DNA molecule Macromolecules that is negatively charged uniformly across the surface so influenced by an electric field intensity voltage and appropriate, we will move to the anode of the electric field The DNA segments will move in the electric field in the gel at a rate inversely... meningococcal vaccine licensed for people older than 55 MPSV4 may be used in people 2–55 years old if the MCV4 vaccines are not available or contraindicated Two meningococcal conjugate vaccines (MCV4) are licensed for use in the U.S The first conjugate vaccine was licensed in 2005, the second in 2010 Conjugate vaccines are the preferred vaccine for people 2 through 55 years of age It is indicated in those with