Nghiên cứu ứng dụng kỹ thuật polymerase chain reaction trong chẩn đoán viêm âm đạo - cổ tử cung do chlamydia trachomatis tại bệnh viện Đại học Y Thái Bình

ĐẠI HỌC QUỐC GIA HÀ NỘI
TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN




Trần Thị Hòa




NGHIÊN CỨU ỨNG DỤNG KỸ THUẬT POLYMERASE
CHAIN REACTION TRONG CHẨN ĐOÁN VIÊM ÂM
ĐẠO - CỔ TỬ CUNG DO Chlammydia trachomatis TẠI
BỆNH VIỆN ĐẠI HỌC Y THÁI BÌNH



Chuyên ngành: Sinh học thực nghiệm
Mã số: 60 42 30





LUẬN VĂN THẠC SĨ KHOA HỌC

NGƯỜI HƯỚNG DẪN KHOA HỌC: GS.TS LƯƠNG XUÂN HIẾN

Hà Nội, Năm 2011

54
MỤC LỤC

MỞ ĐẦU 1
CHƢƠNG 1: TỔNG QUAN 3
Chlamydia trachomatis. 3
Chlamydia trachomatis 6
1. 6
me (enzyme immunoassays  EIAs) 7
- DFA) 8
acid nucleic (nucleic acid amplification tests  NAATs). 8
Chlamydia trachomatis  10
 10
1.3 18
CHƢƠNG 2: ĐỐI TƢỢNG VÀ PHƢƠNG PHÁP NGHIÊN CỨU 23
2.1.  23
2.1.1. 



 23
 23
 23

2.2. 



 23
2.2.1. 



 23
2.2.2.  24
 24
 24
 25
 25
  29
 30
2.2.2.7. 





 Chlamydia trachomatis  ,




 
 31

2.2.2.8. 

, 



31
2.2.3. 





 32

55
CHƢƠNG 3: KẾT QUẢ VÀ BÀN LUẬN 33
3.1. 









Chlamydia trachomatis 33

2

33
 35
 37
 40
 43
 44
3.4.  44
KẾT LUẬN 49
TÀI LIỆU THAM KHẢO 50

56
DANH MỤC BẢNG BIỂU, HÌNH VẼ
Hình 1.1. Cấu trúc hệ gen của vi khuẩn Chlamydia trachomatis…………………… 3
Hình 1.2. Chu kỳ vòng đời của vi khuẩn C.trachomatis……………………………… 4
Hình 1.3. Tỷ lệ nhiễm C.trachomatis ở một số nƣớc Châu Âu……………………10
Hình 3.1. Ảnh điện di kết quả tối ƣu nồng độ MgCl2 với cặp mồi KL1/KL2…….34
Hình 3.2. Ảnh điện di kết quả tối ƣu nồng độ MgCl2 với cặp mồi KL5/KL6…….35
Hình 3.3. Ảnh điện di kết quả tối ƣu nồng độ mồi

KL5/KL6…………………… 36
Hình 3.4. Ảnh điện di kết quả tối ƣu nồng độ mồi

KL1/KL2…………………… 36
Hình 3.5. Ảnh diện di kết quả tối ƣu nhiệt độ gắn mồi KL1/KL2……………… 38
Hình 3.6. Ảnh diện di kết quả tối ƣu nhiệt độ gắn mồi KL5/KL6……………… 38
Hình 3.7. Ảnh diện di kết quả tối ƣu thời gian gắn mồi KL5/KL6……………….39
Hình 3.8. Ảnh diện di kết quả tối ƣu thời gian gắn mồi KL1/KL2…………….….40
Hình 3.9. Ảnh diện di kết quả tối ƣu chu kỳ phản ứng……………………………40
Hình 3.10. Ảnh diện di độ nhạy phản ứng PCR hai lần trên vi khuẩn C. trachomatis……43
Hình 3.11. Kết quả xét nghiệm trên mẫu bệnh phẩm………………………… …45
Bảng 3.1. Thành phần phản ứng PCR lần thứ nhất…………………………… …41
Bảng 3.2. Chu trình nhiệt của phản ứng PCR lần thứ nhất…………………….….42
Bảng 3.3. Thành phần phản ứng PCR lần 2…………………………………….…42
Bảng 3.4. Chu trình nhiệt của phản ứng PCR lần 2……………………………….43
Bảng 3.5. Kết quả xét nghiệm C.trachomatis…………………………………….45
Bảng 3.6. Tỷ lệ nhiễm C.trachomatis theo độ tuổi……………………………… 46
Bảng 3.7. Tỷ lệ nhiễm C.trachomatis theo khu vực……………………………….47

58
BẢNG CHỮ VIẾT TẮT

C. trachomatis
Chlamydia trachomatis
DFA
Direct Fluorescent Antibody
EIAs
Enzyme Immuno Assays
LPS
Lipopolysaccharide
MOMP
Major Outer Membrane Protein
NAATs
Nucleic Acid Amplication Tests
PCR
Polymerase Chain Reaction
TPHA
Treponema Pallidum Hemagglutination Assay



1
MỞ ĐẦU
 
                V
sinh Chlamydia trachomatis 
  Chlamydia trachomatis ,
 












 

 
               
Chlamydia trachomatis  [34]Chlamydia trachomatis hay
 .

 -Chlamydia trachomatis 
 -2005 [15, 18].
Chlamydia
trachomatis 
               Chlamydia
trachomatisChlamydia trachomatis 

[2,9].
 Chlamydia trachomatis 
-   Chlamydia trachomatis. Tuy

.
K NAATs) 


 Polymerase Chain
Reaction PCR) 


2
.  Chlamydia trachomatis
.

 c Nghiên cư
́
u ư
́
ng
dụng kỹ thuật PCR trong chẩn đoán viêm âm đạo, cổ tử cung do Chlamydia
trachomatis tại Bệnh viện Đại học Y Thái Bình 
-              
.
- X

  t 



  




g.

3
Chƣơng 1: TỔNG QUAN
1.1. Vi khuẩn Chlamydia trachomatis.
Chlamydia trachomatis  chi Chlamydia  Chlamydiaceae 
ChlamydiaeChlamydiaeBacteria [2, 9].
 
       , l       
lipopolisaccharide  
  

Chlamydia trachomatis .042.519 nucleotide









.1.  Chlamydia trachomatis
( Theo Deborah Dean et al., (2009) Predicting Phenotype and Emerging Strains
among Chlamydia trachomatis Infections, CDC Home Vol. 15.(9))
Chlamydia trachomatis (C.trachomatis) 
.  
 (Open Reading Frame) 


4
 
 C.trachomatis.
C.trachomatis 5  hu
T -t A, 
 D, E, F, G, H, I, 

[2,9].
C.trachomatis -
[34]. C.trachomatis   .
-    (Elementary Body- EB)        
m
-   (Reticulate Body- RB): Sau kC.trachomatis
     m),  
  -3  
 



  exocytosis).
 -1000   


1.2.  C.trachomatis (theo Yvonne Pannekoek).

5

VC.trachomatis        

   h
C.trachomatis.

C.trachomatis           
 (D,
.
T C.trachomatis
.
Ở phụ nữ
 
 [3] C.trachomatis 


  C.trachomatis             
       C.trachomatis
.
 C.trachomatis 
.  C.trachomatis :
- Tắc vòi trứng ở phụ nữ
               do
C.trachomatis.
- Chửa ngoài tử cung:  do C.trachomatis
 P

thai 9% 
 C.trachomatis .

6
- Viêm vùng chậu: C.trachomatis
. , n

C.trachomatis 
 
- C.trachomatis  (Human papilloma virus)  
.
Ở nam giới
C.trachomatis 
 
. 

.
C.trachomatis 

C.trachomatis 
 C.trachomatis
trong  .
1.2. Các xét nghiệm chẩn đoán vi khuẩn Chlamydia trachomatis [32]
1.2.1. Nuôi cấy
 C.trachomatis  vi
 C.trachomatis 
k 
 cC229.
-  
nhau 
-  
        )         
 
0
C- 5% CO2i

7

  C.trachomatis  
  lipopolysaccharide (LPS) 
 (major outer membrane protein- MOMP).
 
 
 , 
  
 . 
(70-   acid (nucleic acid amplification tests
NAATs) [32].
1.2.2. Kỹ thuật miễn dịch gắn enzyme (enzyme immunoassays– EIAs)
             
C.trachomatis  
 - 
 ng quang

 , C.trachomatis 
    
C.trachomatis     
  e            
C.trachomatis.
 e  

 


 , 
 5%) [32]. 


8
1.2.3. Kỹ thuật kháng thể huỳnh quang trực tiếp (direct fluorescent antibody- DFA)


 MOMP.

       

        
 lam 
,  
C.trachomatisC.trachomatis 

C.trachomatis  

X

. 
             C.trachomatis 
,  
               
NAATs   [32].
1.2.4. Kỹ thuật khuếch đại nucleic acid (nucleic acid amplification tests– NAATs).
NAATs 

 NAATs 
, 
 (trong 1 
 (Polymerase Chain Reaction PCR) 
             C.trachomatis [21]. N


 C.trachomatis

9










 
  
 
C.
trachomatis.   (1985)
[23],  ethidi
  %.
Nested PCR   

khu 
 
 
   
-
do
 Theo  (2002) [22], x

10   (EB) 
C.trachomatis , cho 
.
DNA C.trachomatis. C
PCR 2
 DNA C.trachomatis. Enzym Taq polymerase 
DNA e DNA ch

DNA  
dSau , 


 agarose 1%, 

e








 .
C.trachomatis 
 ), 
.

10


,  
 [13, 17]. 

 uracil- N- 
1.3. Các nghiên cứu về Chlamydia trachomatis trên thế giới và trong nƣớc
1.3



 1.3. hlamydia 
Theo ECDC   [15],    C.trachomatis   
 
 
() 
   
  
  
  trong .
  [20] 



100.000


11

Ng 
  


 ()
  
 l C.trachomatis 

 et al.
  
   C.trachomatis  
 et al.
-  16  20
 


C.trachomatis 

 (Smith et al.
  
  
                
C.trachomatis 

 m C.trachomatis  
3.5% (5/145).     C.trachomatis   .    

 DFAC.trachomatis . et
al. 
 PCR.    

12
 vi khu  
12.C.trachomatis .2%.

C.trachomatis   dao
  .               C.
trachomatis  6%     
 [15].
James B. Mahony  (1992)  C.trachomatis
        [18]
  C.trachomatis . 
      
 C.trachomatis.     trong
    
, PubMed    
  5.1%.  cao   qu    
  (7.9%)   (6.8%),  (6.7%), 15-
19  (6.1%)  20-24  (6.5%).   C.trachomatis   cao 
 .
C.trachomatis  b  
[27].   63.000   C.trachomatis   con
              .   
C.trachomatis  Canada  % m 1997. Tuy , 
        (40% 
70% )   n. 
  trong  C.trachomatis,   hai  ba 
    .    
C.trachomatis   cao ,    nhanh
 .       59  129.5 
  trong khi         ,   263 
100.000.     do    vi
 C.trachomatis .    s 
     . thanh

13
   C.trachomatis    ,      
     ra  15  24 trong  . Trong 
2004,    19   847 100.000.  
1.087  trong  20-24.     
    C.trachomatis cao,       8    
. Nunavut C.trachomatis   (2.520
)    [27].
nested multiplex 
HO1, HO3, Chlam5/Chlam3, KL1/ Gon 5/Gon 3
 C.trachomatis trong 
 [19].   3 
  
    
tham gia. 15-20              
       -
  
 (Sigma  
2 1. 
C   Eppendorf)
        C.trachomatis.      
1
    
agarose 2%,  PCR .2
mM   dNTP; 3 mM MgCl2; 0.5 pmol/l   HO1, HO3, Chlam5 (5'-
CATTATGT-CGGAGTCTG-AGC- ), Chlam3 (5'-GGATGACTCAAGGAATAGT-
CG- 3), 1 l Internal Amplification Control (IAC), 0.4 pmol/l  IAC (5'-
TGTTTGACAGCTTATCAT), 5 l .5  
ion  l /15s, 60/30s, 72/
.2 mM dNTP; 2 mM MgCl2; 0.5 pmol/l 

KL2, Gon5 (5'-GTTCT-TGACGCTCC-ATATCG-    -ACGAGGCAT-
TGAAGCAAAGC- ); 0.4 pmol/l IAC; 1 l      .5 U Taq
 l 94/15s, 58.5/30s,72/30s,

14
  C.trachomatis 
 C.trachomatis .    multiplex PCR 
 C.trachomatis   (20.9%)   .


Farhad B. Hashemi, Babak Pourakbari  [16] 
 -
k Tehran,
 

 


-20
-20 
  r      /3.  /30s, 52/30s,
72 g 25  mmol/l Tris,
pH 8,3; 50 mmol/l KCl; 2.5 mmol/l MgCl2  .1% gelatin); 0.2   
dNTP; 2.5 U Taq    .5 mol/l     -
AACCGTTTTTAATAGTGGCA-   -TTCTGGCCAAGAATTATCC-
   C.trachomatis  17%

 (
 )     C.trachomatis       - 


   
 
  
 [20], 
C.trachomatis cao 
 
 C.trachomatis.  

15


Achchhe L Patel [11]  
            C.trachomatis 
Roche Amplicor test, DFA Trong   (2003
C.trachomatis  30.0%. 
 Surat,   b PACE2 test) 
8.5%,    Ahmedabad,           
C.trachomatis   

  Azerbaijan 3.1%  Bangladesh 3.4%. 
 Manila 23.3%, Cebu, Philippines 37.0%  14.0%  Nicaragoa [11] .
 () [12] 
ELISA  C.trachomatis 
         C.trachomatis    



58  -
..8 
.  

..4 

C.trachomatis 
              
C.trachomatis M
   
d -
5 
 C.trachomatis 
   Chlamydia. cho
    C.trachomatis 

16
 C.trachomatis           
C.trachomatis 
        -TCCGGAGCGAGTACGAAGA-) v
KL2 (5-AATCAATGCCCGGGATT GGT-3). 
25 
 mM MgCl2, 0.28 mM dNTP, 16 pmol  U Taq
polymerase.     
/ ,  . 
. C.trachomatis  
..9% 

C.trachomatis .. [12].
Theo CDC   
 
C.trachomatis 
 
C.trachomatis  

 [15]

 C.trachomatis 

   C.trachomatis 
t.8 ca ;
 . [18].
 C.trachomatis 
ao 


 



17
   
agaro  


 C.trachomatis.
 [22],
 
C.trachomatis v
 
   mM Tris (pH 9,0),
50 mM KCl, 3.5 mM MgCl2, 5          -
TTGCCTTAACCCCACCATT--CGTCCTTCCTAAAAGAG-CTA-
1.25 U Taq 
 


          KL1 (5-TCCGGAGCGAG-
TTACTAAGA-   -ATCAATGCCCGGGATTGGT-   


C.trachomatis 


     
 (EB) 

 
 .5 
theo  
  
C.trachomatis ,  
 


18
C.trachomatis trong , 



1.3.2. Các nghiên cứu trong nước

C.trachomatis  . 

.



Nam [29]. 

39. 
        N.gonorrhoea    
C.trachomatis 

 
C.trachomatis 2.5%.
 
              
-1996 [24].
     

N.gonorrhoeae
             
C.trachomatis  
.4% cho giang mai, 3..8% cho
C.trachomatis, 5.V.

19
  
-  h [3].
  l
danh 
 . 

  C.trachomatis     
C.trachomatis 
 

 C.trachomatis 
C.trachomatis .07%.
C.trachomatis .5%
 [8].
-
s

 [25]
N.gonorrhoea, Smart Check Chlamydia cho
C.trachomatis  0.2%, C.trachomatis 0.6%,
- 
  

 [26]5
C.trachomatis
             C.trachomatis,
C.trachomatis  Lai Chu 2.0%, 1.0%, 27.3%; QuTr 1.0%, 12.9%, 32.7%;
 4.7%, 11.4%, 16.0%; An Giang 7.0%, 10.7%, 11.3%  4.0%,
13.4%, 24.4%.



20
 [29]C.trachomatis 
.5-5%
. Trong
C.trachomatis ..8%.


   [29]

 
  N.gonorrhoea  C.trachomatis 
C.trachomatis/C.trachomatis .9%, 48.4%, 54.9%.
C.trachomatis 
C.trachomatis 
 
 
   .    C.trachomatis       
 
 [1]    
              
C.trachomatis.
NC.trachomatis 
. 

20 [31]
C.trachomatis 
C.trachomatis 
 C.trachomatis 

. 
C.trachomatis 

