I: INTRODUCTION

1. Tuberculosis and Mycobacterium tuberculosis ?

a, Tuberculosis
Tuberculosis is an infectious disease usually caused by the bacterium
Mycobacterium tuberculosis. The disease mainly affects the lungs but can also
affect other parts of the body.
Symptoms : persistent cough with bloody mucus, fever, night sweats, and
weight loss.
b, Mycobacterium tuberculosis
Mycobacterium tuberculosis belong to the family Mycobacteriaceae, 3–5
μm long, hairless, rounded at both ends, seeded, and stand singly or in clusters on
Ziehl-Neelsen staining slides.
* Characteristics:
- Resistant to alcohol and acid, Complete animosity
- Slow growing, only spawns once every 20-24 hours
- Resistant to drugs to treat pulmonary tuberculosis, lymph node tuberculosis,
peritoneal tuberculosis…
* The viability of TB bacteria
-Under the sun, TB bacteria are killed after 1.5 hours
-TB bacteria stop growing at 42°C and die after 10 minutes at 100°C
-Alcohol 90°C can kill TB bacteria in 3 minutes
-In 5% phenic acid, bacteria can only live for about 1 minute
* Current method for detecting TB
The BACTEC MGIT 960 system is a noninvasive, nonradiometric system that
exploits the fluorescence of an oxygen sensor to detect growth of mycobacteria in
culture. A ruthenium pentahydrate oxygen sensor embedded in silicon at the
bottom of a tube containing 8 ml of modified Middlebrook 7H9 broth fluoresces
following the oxygen reduction induced by aerobically metabolizing bacteria
within the medium. A compact instrument incubates and tests, according to
onboard algorithms, up to 960 culture tubes (nomenclature)
2. Pyrosequencing?
Pyrosequencing is a method of DNA sequencing that differs from Sanger
sequencing, in that it relies on the detection of pyrophosphate release and the
generation of light on nucleotide incorporation, rather than chain termination
with dideoxynucleotides.
II, MATERIALS AND METHODS
I. Materials
Sputum sample colected from retreatment pulmonary tuberculosis (PTB)
patient.
For decontamination and preperation of sputum sample:
+) 2% NaOH
+) 0,5% NACLC
+) Phosphate buffer (pH=6,8)
 +) 0,9% NaCl
For PCR amplification:
+) 0.5 μl 10 × PCR buffer.
+) 0.2 mM deoxynucleotide triphosphates
+) 200 nM of each primer
+) 10 ng of DNA.
For conducting pyrosequencing:
+) streptavidincoated sepharose beads
+) PyroMark Gold Q96 SQA reagents
For detection of M.tuberculosis by the Bactec MGIT 960 system:
+) RIF: 1.0 µg/ml
+) INH: 0.1 µg/ml
+)SM: 1.0 µg/ml
+) EMB: 5.0 µg/ml
+) OFL: 2.0 µg/ml
+) AMK: 1.0 µg/ml
II. Methods
* Ethics considerations (Đạo đức khoa học)
The informed consent was obtained from each patient who was treated in
accordance with the Helsinki Declaration on the participation of human subjects
in medical research. (các đối tượng thí nghiệm đc chọn lọc tuân theo tuyên bố
Helsinki đảm bảo nguyên tắc đạo đức trong nghiên cứu) 

a, Source of clinical isolates and sputum samples (Nguồn phân lập lâm sàng
và mẫu đờm (dịch họng))  
- A total of 205 clinical M.tuberculosis isolates and 24 sputum samples were
obtained from retreatment pulmonary tuberculosis (PTB) patients in Shanghai
Pulmonary Hospital from January 2010 to March 2011. (Tổng số 205 phân lập
M.tuber tuberculosis lâm sàng và 24 mẫu đờm được lấy từ các bệnh nhân tái điều
trị PTB tại Bệnh viện phổi Thượng Hải từ tháng 1 năm 2010 đến tháng 3 2011)  
- All of the samples were identified as M. tuberculosis by Bactec MGIT 960
culture, Ziehl-Neelsen staining and standard biochemical identification testing
(Tất cả các mẫu đều được xác định là M. tuberculosis bằng phương pháp nuôi
cấy Bactec MGIT 960, nhuộm Ziehl-Neelsen, và kiểm tra nhận dạng sinh hóa
tiêu chuẩn)  
b, Specimen processing and genomic DNA extraction (Xử lý mẫu vật và tách
chiết DNA bộ gen) 
- For sputum samples, prior to DNA extraction, each sputum sample was
decontaminated with an equal volume of 2% NaOH and 0.5% NALC, incubated
at room temperature for 20 min, neutralized with 67 mM phosphate buffer (pH
6.8) and then centrifuged at 3,300 × g for 20 min at 4°C. (Đối với các mẫu đờm,
trước khi tách chiết DNA, mỗi mẫu đờm được khử nhiễm bằng một thể tích bằng
NaOH 2% và 0,5% NALC, ủ ở nhiệt độ phòng trong 20 phút, được trung hòa
bằng đệm phosphat 67 mM (pH 6,8) và sau đó ly tâm ở 3,300 × g trong 20 phút ở
4 ° C.)  
- The processed sediment was washed once using a sterile 0.9% NaCl solution
and re-suspended in 1.0 ml sterile 0.9% NaCl solution. (Cặn đã xử lý được rửa
một lần bằng dung dịch NaCl 0,9% vô trùng và được hòa tan (?) lại trong 1,0 ml
dung dịch NaCl 0,9% vô trùng.)  
- The 500 μl aliquots for the DNA extraction were inactivated and lysed for 30
min at 85°C and DNA extraction was performed as described above for the
extraction of clinical isolates previously (500 μl mẫu để tách chiết DNA đã bị bất
hoạt và được ly giải trong 30 phút ở 85 ° C và quá trình chiết xuất DNA được
thực hiện như mô tả ở trên để tách chiết các dòng phân lập lâm sàng trước đó (là
tách chiết như bình thường?))  
c, Design of primers and PCR amplification (Thiết kế mồi và khuếch đại
PCR)  
- Regions of the rpoB, katG, embB, rrs, gyrA and rpsl genes were amplified by
conventional PCR (Các vùng gen rpoB, katG, embB, rrs, gyrA và rpsl được
khuếch đại bằng PCR thông thường) 
- Amplification and sequencing primers for pyrosequencing were designed with
PSQ assay design software (Các mồi khuếch đại và giải trình tự được thiết kế
bằng phần mềm thiết kế PSQ)  
- Chemicals for PCR: 50 μl consisting of 0.5 μl 10 × PCR buffer, 0.2 mM
deoxynucleotide triphosphates (Takara), 1U Taq DNA polymerase (Takara), 200
nM of each primer, and 10 ng of DNA. (50 μl bao gồm 0,5 μl 10 × PCR đệm, 0,2
mM deoxynucleotide triphosphat (Takara), 1U Taq DNA polymerase (Takara),
200 nM mỗi mồi và 10 ng DNA. 
- Thermo-cycling conditions were as follows: 95°C for 3 min followed by 50
cycles of 30 s at 95°C, 30 s at respective annealing temperature, 30 s at 72°C and
final elongation at 72°C for 10 min (Điều kiện chu kỳ nhiệt như sau: 95 ° C trong
3 phút tiếp theo là 50 chu kỳ 30 s ở 95 ° C, 30 s ở nhiệt độ ủ tương ứng, 30 s ở 72
° C và cuối cùng kéo dài ở 72 ° C trong 10 phút.) 
Table 1 Primers and thermo-cycling conditions of the pyrosequencing assays
Thiết kế và nhiệt độ lai primer. (Từ trái sang: Thuốc kháng sinh chỉ định, gene
đb quy định tính kháng thuốc, chuỗi gene, nhiệt độ lai, kích thước, loci mục tiêu)

d, Protocols of pyrosequencing (Quy trình pyrosequencing) 

- The results from pyrosequencing were compared with the gold standard
“Sanger sequencing” (các kết quả từ giải trình tự pyrose được so sánh với tiêu
chuẩn vàng "giải trình tự Sanger" (Đối chứng độ chính xác)). The
pyrosequencing results were considered valid if the pyrosequencing results were
consistent with Sanger Sequencing results. (Kết quả pyrosequencing được coi là
hợp lệ nếu kết quả phù hợp với kết quả giải trình tự Sanger.
- Pyrosequencing was performed using PyroMark 96MA instrument. PSQ 96MA
system had two modes to detect gene mutation. BOTH are used.
+) dedicated sequence analysis (SQA) Software which could give true sequence
information about 50 bases.
+) single-nucleotide polymorphism (SNP) Software which could support fast
genotyping results.
2 chế độ pyrosequencing của kit
+) SQA trình tự chính xác vs mẫu 50bp
+) SNP đưa ra kết quả xác định trình tự nhanh chóng với độ chính xác cụ thể
từng nu 1
- Firstly the PCR amplification product immobilized on the streptavidincoated
sepharose beads was converted into single-stranded DNA template and
purified with a vacuum preparation tool. (sản phẩm khuếch đại PCR trên hạt
sepharose phủ streptavidin, được chuyển đổi thành khuôn mẫu DNA sợi đơn)
- Then a sequencing primer was subsequently annealed to the template.
Pyrosequencing was performed in an automated Pyro- Mark 96MA instrument
according to the manufacturer’s instructions. (gắn primer)
- For the SQA mode detection, a PyroMark Gold Q96 SQA reagents and cyclic
dispensation of the nucleotide was used
- For the SNP mode, the genotyping results were directly obtained by using the
PyroMark Gold Q96 reagents.
- Amplified H37Rv genomic DNA was employed as a control. Đối chứng
- Comparison with conventional drug susceptibility testing DST was performed
using the Bactec MGIT 960 mycobacterial detection system. Drug concentrations
used in the MGIT 960 system used are listed above. (phương pháp pyrose được
dùng để so sánh phương pháp truyền thống Bactec trong thí nghiệm này)

Table 2 Optimum conditions for detection mutation of rpoB, katG, gyrA,

rrs, embB and rpsl genes by pyrosequencing.
Chế độ tối ưu để xác định gene
III. Explanation for usage of Bactec and Sanger control:
1. Comaperison to Bactec MGIT 960 mycobacterial detection system:
The mutations of rpoB and gyrA gene were detected by pyrosequencig with
the SQA mode, and the mutations of katG, rpsl, embB, gyrA and rrs gene
were detected by pyrosequencing with SNP mode. Compared with the
Bactec MGIT 960 mycobacterial detection system, the accuracy of
pyrosequencing for the detection of RIF, INH, EMB, SM, AMK and OFL
resistance in clinical isolates. This is used as a baseline comparison to infer
the effectiveness of pyrosequencing when compare to the traditional method
of Bactec MGIT 960 kit.
2. Comparison to Sanger Sequencing:
10 collected M.tuberculosis clinical isolates with known sequence by Sanger
Sequencing were detected gene mutation by pyrosequencing with both SQA
 mode and SNP mode. Then the results from pyrosequencing were compared
with the gold standard “Sanger sequencing” results by the Identifire
software. The pyrosequencing results were considered valid if the
pyrosequencing results were consistent with Sanger Sequencing results. By
doing this we can infer whether or not the test results from pyrosequrncing is
more acurate, consuming less time and reagent.
IV. RESULT:
DST using Bactec MGIT 960 system DST of RIF, INH, SM, EMB, OFL and
AMK was detected by Bactec MGI 960 system. Of the 229 retreatment PTB
patients samples, 89.5% cases were identified resistant to at least one of the
tested drugs, above 50% cases were resistant to four or five tested drugs and
6.6% cases were resistant to only one drug (bưng y vô slide, phần này về kết
quả của kit Bactec)
Compared with Bactec MGIT 960 method, pyrosequencing showed higher
sensitivity and specificity in RIF, OFL, AMK and SM than in INH and
EMB. The detection of EMB resistance showed the lowest sensitivity among
six
drugs not only in clinical isolates (58.7%) but also in sputum sample
(41.7%). The INH and AMK showed the highest specificity (100%) in all the
detecting drugs and samples. These results were present in Table 5. (bưng y
vô slde phần này kết quả của pyros so sánh vs Bactec)
 Table 3 Mutations of the rpoB gene identified by
pyrosequencing in clinical isolates and sputum samples
Các đột biến gene rpoB quan sát được (lý do quan tâm rpoB cụ thể do gen đa
hình polymorphic với nhiều kiểu gen khác nhau và khả năng kháng thuốc
RIF (rifampicin))

Table 4 Mutations of the katG, embB, rrs, gyrA and rpsl

genes identified by pyrosequencing in clinical isolates
and sputum samples
Đột biến các gene katG, embB, rrs, gyrA và rpsl
Cách đọc bảng trái sang tên thuốc kháng sinh, tên gene đb, aa bị thay đổi và
vị trí, codon đb, kết quả đb dựa trên Bactec.

Table 5 Comparison of pyrosequencing and Bactec MGIT 960 DST

So sánh kết quả với Bactec MGIT 960

In the pyrosequencingoperating system,two modes, SQA and SNP, could be

used, but the two modes differed in several aspects. For example, SQA mode
detected a short and medium length DNA fragment, and SNP mode detected only
a single base variation. In our experiments, although SQA mode could detect all
6 drugs resistance, we chose the SNP mode rather than SQA mode to detect the
 mutation of katG, rpsl, embB and rrs based on the below reasons. By SNP mode,
it was possible to sequence 96 samples in approximately 15 min, while it needed
70 min by the SQA mode. However, for rpoB and gyrA gene, we chose the SQA
mode to detect the mutation because the sequence of interesting had been highly
polymorphic and a short region should be sequenced. (do các gene trong thí
ngiệm chủ yếu đột biến điểm nên chế độ SNP phù hợp hơn do có thể xđ chính xác
khác biệt từng nu riêng lẻ và ít tốn tg và nguyên liệu hơn còn SQA phù hợp hơn
khi các gene polyphormic)
Pyrosequencingcan be used as a practical molecular diagnostic tool for screening
and predicting the resistance for re-treatment pulmonary tuberculosis patients.
Kết luận cho quan trọng bưng vô slide
V. COMPARISON:
Characteristics Pyrosequencing Sanger sequencing
Principles Sequencing during Synthesize chains with a
synthesis (sequencing by difference of 1 nucleotide and
Synthesis) by detecting the then electrophoresis on
light generated when PPi capillary electrophoresis (ABI
released by a dNTP being or Becman Coulter)
incorporated.
Sample Only put the purified PCR PCR products after
preparation product into the sequencing purification need to run a
machine with real-time second round of PCR, purified
results then electrophoresis on the
sequencing machine
Wattage 24 sample or 96 sample/ 1 8 sample or 24 sample/1 run
run
Sensitivity <2% mutated sequence and > 25% mutant sequence
>98% Wt
Application Detection of mutations, Detect known mutations or
identification of bacteria, find new mutations,
viruses, resistance identification of bacteria,
mutations, high sensitivity viruses, segment analysis (eg
when studying SNPs, STR - Bloodline, Florescent
Quantification of PCR...)
Methylation