MOLECULAR CHARACTERIZATION OF NEISSERIA MENINGITIDIS STRAIN CIRCULATING IN THE NORTH OF VIETNAM

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.......
1


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

2


CONTENTS

3


TABLE OF CONTENTS

FIGURE OF CONTENTS

4


LIST OF ACRONYMS
CSF: cerebrospinal fluid
N.meningitidis: Neisseria meningitidis
PCR: Polymerase Chain Reaction

5


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 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
6

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.

7


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
8

between 10 and 20 percent.[1] The incidence of endemic meningococcal disease during
the last 13 years ranges from 1 to 5 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
9


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 9 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 8 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


Isolated from a Patient with bacterial 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 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 impaired immunity, such as nephrotic syndrome or splenectomy. The Centers
for Disease Control and Prevention (CDC) publishes information about who should
receive meningococcal vaccine.[9]
In June 2012, the U.S. Food and Drug Administration (FDA) approved a combination
vaccine against two types of meningococcal diseases and Hib disease for infants and
children 6 weeks to 18 months old. The vaccine, Menhibrix, was designed to prevent
disease caused by Neisseria 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 African meningitis belt. It was made by generic drug maker Serum Institute of
India and cost 50 U.S. cents per injection. Beginning in Burkina Faso in 2010, it has
been given to 215 million people across Benin, Cameroon, Chad, Ivory
Coast, Ethiopia, Ghana, Mali, Niger,Mauritania, Nigeria, Senegal, Sudan, Togo and Gam
bia.[12] The vaccination campaign has resulted in near-elimination of 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
27.4% to 34.7% (Oberti, Hoi et al. 1981). Seventy percent of cases occur in children
aged 3 to 15 years old. After the large epidemic of meningitis in 1977, four researchers
tracked over time for meningitis was conducted from 1993 to 2005 (US, Diep et al.,
1998; Tran, Le et al. 1998; DD 2006 ; Nguyen, Tran et al. 2007). In particular,
meningococcal meningitis accounted for 0.5% of positive cases confirmed by culture
method from blood samples and about 4 to 8.5% of cases of bacterial meningitis was the
cause determined. From 2000 to 2002, the estimated incidence of meningococcal
meningitis in Hanoi, Vietnam, is 21.8 cases / 100,000 children aged 7-11 months (95%
CI 5.0-94.4) and 2.6 cases / 100,000 children under 5 years of age (95% CI 0.8-8.5) (DD
2006). However, no data on the disease-causing serogroups in this study. Full study on
the most recent meningococcal meningitis and Neisseria meningitidis infection in
children in Vietnam was published by Kim et al (Kim, Kim et al. 2012) according to a
survey on 700 samples collected children under age 5 in Hanoi, using PCR specificity,
detecting N. meningitidis infection rate in this group was 14.2%. According to our
understanding, so far no study has been conducted to assess the genetic variation or
genes regulated virulence of pathogenic serogroups of N. meningitidis in Vietnam's
2.3 Biological Characteristics
2.3.1 The nomenclature and classification of Neisseria meningitidis
•

Nomenclature

Kingdom: Bacteria
Phylum: Proteobacteria
Class: Beta Proteobacteria

Family: Neisseriaceae

Genus: Nesseria
Species: N. meningitides

Ordo: Neisseriales

Serogroup: A, B, C, D, 29E, H,
I,L,W135,X,Y,Z

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Genus

Specific

Gram

species

stain

Shape

Capsule Arrange Mobile Respira--Grow
-tory

Neisseria N.gonorrhoeae Gram (-) Hình hạt Tạo vỏ

Culture

Inside /
outside cell

Phế

Không Hiếu

Thayer- Gonococcus:
martin

Neisseria

cà phê, hoặc

cầu

di

meningitides

hai mặt không

khuẩn

động

khí

inside cell

Blood

Neisseria

dẹt đối

Agar or

meningitidis:

xứng

Chocolate out cell.

nhau

Table 2.1. Biology characteristic of Neisseria meningitidis
Meningococcal is Gram-negative cocci, size changes, can be found in the form the
lonely or beans with flat sides facing each other and can be located inside or outside of a
neutrophil, hairless, no spores, most strains are capsule.

Figure 2.2 : Gram stain of Neisseria meningitidis in cerebrospinal fluid (CSF) with
associated PMNs.
Polysaccharide layer (PS) capsule: as antigens, that generated antibodies
protection (except for group B). Based on the heterogeneity of the structure and
13


properties of PS antigen was found to be 13 serogroups of meningococcal bacteria
including: A, B, C, D, 29E, H, I, K, L , W135, X, Y, Z. Mannosamine serogroup A

contains phosphate, while serogroups B, C, Y, W135 contains sialic acid - which plays
an important role in the survival and toxicity. Capsular polysaccharide of serogroup B
and C include acid homopolymer of N - acetyl - neuraminic associated with α – 2.8 and
α - 2.9. Small differences in the structure leads to different immne properties explicitly:
while structural α - 2.9 is a strong antigen to the body and produce antibodies that
protect the α - 2, 8 has antigenically very weak.
The structure of the group antigen and subtypes in serum change in vivo quickly
on an individual person and in the community. The loop (circuit) can be changed on the
exposed surfaces of both porins, can change antigenically by adding or subtracting the
amino-acid or by horizontal transfer of gene fragments representing parts.
X antigens: antigens with Neisseria Gonorrhoeae, Streptococcus Pneumoniae. This
antigen is used in a number of serological diagnostic techniques.
2.3.2 Culture medium
Meningococcal bacteria are aerobic, developing in an environment with 5% blood
agar, Thayer-Martin agar environmental or chocolate, even Luria Bertani medium, at a
temperature of 35-37, in 5-7% CO 2. On blood agar, small colonies, round, opaque,
convex, whitish-gray, non-hemolytic, 1- 3 mm in diameter. When using implants
pushing, sliding easily colonies on the agar.
Distinguish meningococcal bacteria normally be based on the shape, the result
Gram stain, biochemical tests: oxidase and catalase reaction positive, ferment glucose,
maltose, but not sucrose or lactose fermentation.

14


Hình 2.3 :Khuẩn lạc Neisseria meningitidis trên thạch chocolate. [14].

Hình 2.4: Khuẩn lạc Neisseria meningitidis trên 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 4 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 thanh (VR1;VR2;VR3)

16


Polysascharide capsule

Phenotype (B:NT:NT/P1.4/NT)
PCR phát hiện Nm
(CtrA;CrgA).
Nhóm huyết thanh:
- SiaD (B;C;Y;W135)
- mynB (A)
Dịch tễ học phân tử:
MLST; PorA;fetA

PorB (lớp 2 hoặc 3 OMP)
Nhóm huyết thanh
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 4 capsule operon: 01 of which
encode synthetic capsule (called syn or sia gene, depending upon the nomenclature
system used) and 03 for 4 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 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 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 8 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), 3 [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 4 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 này nên chưa biết được 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 1. Grow colonies overnight at 37°C in shake cabinet with 5ml Luria- Bertani fluid
medium.
Step 2. Pour 1,5ml bacterium fluid overnight above into eppendorf 1,5ml.
Step 3. Centrifuge at 10,000 rpm and 4ᵒC for 5 min
Step 4. cellular biomass collected by pouring off supernatant.
Step 5. Add 400µl Lysis buffer
Step 6. Add K protein that finally concentration of K protein is 20ng/ml. Incubate at 55
about 2 hours in a recirculating water bath. Vortex the well mixture briefly before
incubate.
Step 7. Add 400µl phenol: clorophorm: isoamyl (25:24:1). Vortex the well about 10
seconds. Để yên 10 minitues.
Step 8. Centrifuge at 12000 rpm and 4 for 20 minutes.
Step 9. Add 400µl isopropanol aim to precipitate DNA. Then, incubate for about 1
hour in a recirculating water bath.
Step10. Centrifuge at 12000 rpm and 4 for 20 minutes to take DNA precipate.
Step11. Pour off supernatant, washing by alchol 70. After centrifuge 1000rpm and 4 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, ở phần này cũng vậy ạ, em đang chờ đến bước
này, rồi Thầy Quyền sẽ thiết kế mồi. Sau đó, em mới hỏi được xem Thầy Quyền dùng
phương pháp, phần mềm nào 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

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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
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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.

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