KULLANICI KILAVUZU
Ampliquality
MYC-TE
Code 03-18A Code 03-18R
Kit for the screening and typing
of Tubercular and Atypical Mycobacteria
1. PRODUCT INFORMATION 3
2. KIT CONTENTS 4
3. STORAGE AND STABILITY OF THE REAGENTS 5
4. PRECAUTIONS FOR USE 5
5. SAFETY RULES 7
5.1. General safety rules 7
5.2. Safety rules about the kit 8
6. MATERIALS REQUIRED, BUT NOT PROVIDED 9
6.1. Reagents 9
6.2. Instruments 9
6.3. Materials 9
7. PREPARATION OF REAGENTS 10
8. INTRODUCTION 11
9. TEST PRINCIPLE 13
10. PRODUCT DESCRIPTION 13
11. COLLECTION, MANIPULATION, AND PRE-TREATMENT OF SAMPLES 14
11.1. Respiratory specimens 15
11.2. Histological samples 15
11.3. Blood 16
11.4. Cerebral fluid 16
12. SCREENING PROTOCOL 17
12.1. DNA extraction 17
12.2. DNA amplification 18
12.2.1. TST DNA amplification 18 12.2.2. Amplification of Mycobacterium DNA 18
12.3. Visualization of amplification products 19
12.3.1. Agarose gel electrophoresis 19
12.3.2. Sample loading 19
12.3.3. Interpretation of results: TST and Mycobacterium DNA amplification 20
13. TYPING PROTOCOL 21 13.1. Typing by digestion with restriction enzymes (RLFP) 21 13.2. Visualization of restriction products 21 13.2.1. High resolution agarose gel electrophoresis (HR) 21
13.2.2. Sample loading 21
13.2.3. Interpretation of typing results 22
14. TROUBLESHOOTING 25
15. DEVICE LIMITS 27
16. DEVICE PERFORMANCES 27
16.1. Specificity 27
16.2. Diagnostic significance 27
16.3. Diagnostic and analytic sensitivity 27
17. REFERENCES 28
17.1. Web sites 28
1. ÜRÜN BİLGİSİ
Var olan kılavuz aşağıdaki ürünler içindir:
MYC-TE (Code 03-18A)
Kit for screening and typing of Tubercular and Atypical Mycobacteria, by DNA amplification of the 65-KDa protein region and RFLP analysis. It contains all the reagents for the amplification and visualization of the products by agarose gel electrophoresis.
Kod Ürün Pkg
03-18A-25 MYC-TE 25 tests
03-18A-50 MYC-TE 50 tests
MYC-TE-amplification reagents (Code 03-18R)
Kit for screening and typing of Tubercular and Atypical Mycobacteria, by DNA amplification of the 65-KDa protein region and RFLP analysis..
Kod Ürün Pkg
02-10R-25 MYC-TE – amplification reagents 25 tests 03-18R-50 MYC-TE – amplification reagents 50 tests
2. KİT İÇERİĞİ
Warning: kits with different codes correspond to different components.
(kısaltma: X= kit içeriğinde bulunan komponent; 0= kit içeriğinde bulunmayan komponent)
KUTU P
– 20°C DE SAKLANIR
code 03-18A code 03-18R
AÇIKLAMA ETİKET
COLOU R OF TUBE (T)
OR LID
25 tests 50 tests 8 tests
X X Single dose premix MYC
tubes Colourle
ss (T) 25 50 8
X X
Single dose premix TST (thiosulfate sulfurtransferase -rhodanese) tubes
Blue (T) 25 50 8
X X Taq DNA polymerase AB TAQ
5 U/μL Red 50 μL 100 μL 1 x 12 μL X X Restriction enzyme 1 Enzyme 1 Green 1 x 45 μL 1 x 82,5 μL 1 x 19,5 μL X X 10X Reaction buffer for
restriction enzyme 1 10X Buffer
for enzyme 1 Green 1 x 90 μL 1 x 165 μL 1 x 40 μL X X Restriction enzyme 2 Enzyme 2 Violet 1 x 15 μL 1 x 27,5 μL 1 x 6,5 μL X X 10X Reaction buffer for
restriction enzyme 2 10X Buffer
for enzyme 2 Violet 1 x 90 μL 1 x 165 μL 1 x 40 μL 0 X Mineral oil* Mineral oil 1 X 2 mL 1 X 3 mL 1 X 800 μL
KUTU F
+2°/ +8°C DE SAKLANIR
code 03-18A code 03-18R
AÇIKLAMA ETİKET
COLOU R OF TUBE (T)
OR LID
25 tests 50 tests 8 tests
X 0 Loading buffer for
electrophoresis 6X Blue Blue 1 x 375 μL 1 x 750 μL 1 x 120 μL
KÜÇÜK ÇANTA
– 20°C DE SAKLANIR
code 03-18A code 03-18R
AÇIKLAMA ETİKET
COLOUR OF TUBE
(T) OR LID
25 tests 50 tests 8 tests
X X Plasmidic DNA containing part of mycobacteria genome
Positive control
MYC-DNA Blue 1 x 60 μL 1 x 60μL 1 x 20 μL
KUTU A
+15°/ +25°C DE SAKLANIR
code 03-18A code 03-18R
AÇIKLAMA ETİKET
COLOU R OF TUBE (T) OR LID
25 tests 50 tests 8 tests
X 0 Agarose molecular biology
grade AGAROSE 1 x 14 g 1 x 26 g 1 x 5 g
X 0 Agarose for high resolution
electrophoresis HR
AGAROSE 1 x 14 g 1 x 26 g 1 x 5 g X 0 Electrophoresis buffer
TRIS-Acetate-EDTA pH: 8,00 50X TAE 1 x 85 mL 1 x 170 mL 1 x 30 mL X 0 Mineral oil Mineral oil 1 x 2 mL 1 x 3 mL 1 x 800 μL
* = The mineral oil is in both 03-18A and 03-18R kit. However, for transport reasons, in the first case the reagent is in box A, while in the second case, it is in the manual’s bag. In both cases, the mineral olio is stored at room temperature.
3. REAKTİFLERİ SAKLAMA KOŞULLARI
Kitin herbir komponenti, tekli kutuların etiketlerinde belirtilen talimatlara gore saklanmalıdır.
Özellikle:
KUTU P -20°C de saklanır
Küçük çanta -20°C de saklanır
KUTU F +2°/ +8°C DE SAKLANIR
KUTU A +15°/ +25°C DE
SAKLANIR
Önerilen sıcaklıkta saklandığında, tüm test reaktifleri son kullanım tarihine kadar stabil kalır.
4. KULLANIM İÇİN ÖNLEMLER
• Kit; moleküler biyoloji tekniklerini diagnostikte uygulama eğitimi almış ve tecrübesi olan araştırmacı tarafından kullanılmalıdır;
• Kit prosedürüne başlamadan önce kullanım kılavuzunu dikkatlice ve tamamen okuyun;
• Ürünleri ısı kaynağından uzakta tutun;
• Son kullanım tarihi geçmiş kitparçalarını kullanmayın;
Saklama koşulları, kutu bütünlüğü ya da metod uygulaması ile ilgili herhangi bir şüpheniz olduğunda, AB ANALITICA teknik destek ile bağlantı kurun:
laboratorio@abanalitica.it .
Nükleik asitlerin amlifikasyonunda, araştırmacı aşağıdaki özel önlemleri almalıdır:
• Filtreli uç kullanın;
• Biyolojik örnekler, ekstrakte edilmiş DNA, kit içeriğindeki pozitif kontrol ve tüm amplifikasyon ürünleri, amplifikasyon reaktiflerinden farklı bir yerde saklanmalıdır.
• PCR öncesi ve sonrası için farklı alanlar hazırlayın ve iki alan arasında materyal (pipetler, uçlar, tüpler, vs.) paylaşımı yapmayın.
• Sık sık eldiven değiştirin.
• Benç yüzeyini %5 sodyum hipoklorit ile temizleyin.
• Kullanımdan önce reaktifleri oda ısısında eritin. Taq polymerase ve ekstrakte DNA yı hızlı bir şekilde oda ısısında ve buz-kalıbı üzerinde ekleyin.
5. GÜVENLİK KURALLARI
5.1. Genel Güvenlik Kuralları
• Reaktifler ve klinik örnekler ile çalışırken tek kullanımlık eldiven kullanın ve çalışma bitiminde ellerinizi yıkayın.
• Ağızla pipetleme yapmayın.
• Bilinen hiçbir diagnostik metod enfeksiyon ajanlarının yokolduğunun garantisini vermediğinden, herbir klinik örneğin potansiyel enfeksiyonlu olduğu göz önünde bulundurulup, bu şekilde çalışılması gereklidir.
• Klinik örnekler ile direk temasta olan cihazların kontaminasyonlu olduğu ve bu şekilde kullanılması gerektiği göz önüne alınmalıdır. Örneklerin kazara etrafa dökülmesi durumunda, %10 Sodyum Hipoklorit ile temizleyin. Temizlenmiş materyaller, kontamine ürünler için olan özel konteynerlere atılmalıdır.
• Klinik örnekler, materyaller ve kontamine ürünler aşağıdaki dekontaminasyondan sonra atılmalıdır:
30 dakika %5 Sodyum Hipoklorit (1 volüm %5 Sodyum Hipoklorit solusyonu herbir 10 volüm kontaminasyonlu sıvı için) solusyonuna batırılmalı
YA DA
en az 2 saat 121°C de otoklavlanmalı (NOT: Sodyum Hipoklorit içeren solusyonları otoklavlamayın !!)
5.2. Kit Hakkında Güvenlik Kuralları
Bu kit kullanımı için riskler tekli komponentler ile ilişkilidir:
Tehlikeli Komponentler:
ETHIDIUM BROMIDE
3,8-diamino-1-ethyl-6-phenylphenantridiumbromide <2%
Risk tanımı: T (Toksik)
RİSK İBARESİ VE S İBARESİ
R 23 ve R 68 İnhalasyon için toksik. Geri dönülmez etkiler riski.
S 36/37 45 Laboratuar giysisi ve tek kullanımlık eldiven kullanın.
Kaza veya rahatsızlık durumlarında, doktora başvurun ve paket etiketini gösterin.
R ve S ibareleri kit ile sağlanan konsantre ürünlere işaret etmektedir.
Özellikle Ethidium Bromide için, agaroz jelde dilusyona kadar.
Konsantre Ethidium Bromide uygulamasında kimyasal dağıtıcı buharlı kabin kullanın. Seyreltilmiş Ethidium solusyonu ile çalışırken daima laboratuar giysisi ve tek kullanımlık eldiven giyin.
Bu ürünler genel çöplere atılamaz. Kurutucu system kullanılmamalıdır. Atılım için yasal kuralları uygulayın.
Ethidium Bromide kaza ile dökülmesi durumunda Sodyum Hipoklorit ve su ile temizleyin.
İstenildiğinde ürünlerin Güvenlik data sheet (MSDS) gönderilebilir.
6. KİT İÇERİĞİNDE BULUNMAYAN GEREKLİ MATERYALLER
6.1. Reaktifler
• Reagents for DNA extraction;
• Steril DNase ve RNase free su;
• Distile su;
• Reagents for the visualization of products by agarose gel electrophorisis (necessary for the 03-18R kit)
6.2. Cihazlar
• Laminar flow kabini (kontaminasyondan korunmak için amplifikasyon premikse TAQ polymerase eklenirken kullanılması önerilir; ekstrakte DNa eklenirken başka bir laminar flow kabini kullanılması önerilebilir);
• Mikropipetler (aralık: 0,22 µL; 0,510 µL; 220 µL);
• Thermalcycler;
• Tartı;
• Manyetik ısıtıcı ya da mikrodalga;
• Mikrosantrifüj (max 12-14.000 rpm);
• Kimyasal kabin (Ethidium Bromide uygulamasında kullanılması önerilir);
• Agaroz minijel için yatay elektroforez haznesi;
• Güç kaynağı (50-150 V);
• UV Transilluminator;
• Foto kamera ya da imaj analizör.
6.3. Materyaller
• Tek kullanımlık eldivenler;
• Tek kullanımlık filtreli uçlar (aralık: 0,2-2 µL; 0,5-10 µL; 2-20 µL);
• TBE dilusyonu için dereceli silindirler (1L);
• Agaroz jel hazırlamak için pyrex şişe ya da Becker;
• Parafilm.
• Sterile microtubes (0.2-0.5mL) for restriction reaction.
7. PREPARATION OF REAGENTS
To make 1 L of TAE Buffer (1X):
20 mL 50X TAE ile 980 mL distile suyu karıştırın.
8. GİRİŞ
Mycobacteria are gram-positive bacilli, alchool-acid resistant, asporigenic, strictly aerobics or microaerophilic, motionless. The Mycobacterium genere encompasses the Mycobacterium tubercolosis complex and more than 80 species of non tubercular mycobacteria (atypical) which include pathogen, opportunistic, and non pathogen species.
The most important species of the Mycobacterium genus are the M.
tuberculosis, the aetiological agent of tuberculosis, one of the most important infectious diseases in the world, which accounts for 8 millions new cases and for more than 2 millions of deaths every year (Dye et al., 1999). The most diffused and known form of tuberculosis is the pulmonary disease, even if, almost each organ can be involved.
The nontubercular Mycobacteria, instead, (MOTT, Mycobacteria other than tuberculosis) tend to give pseudo-skin infections, that in man can be the cause of sporadic pathologies (meningitis, lymphoadenopaties, bronchopneumopathologies, genital-urinary infections, skin infections, infections of the soft tissues, bones, and joints) (Wayne et al., 1992).
Among the different atypical species, the most abundant clinical samples are M. avium and M. intracellulare. The infections given by those ubiquitous Mycobacteria (water, air, soil) can be clinically significant especially if they are associated with acquired immunodeficiency disease (solitary pulmonary nodules and chronic bronchitis, bronchiectasis) (Horsburgh, 1991).
This complex (MAC) is considered as an intracellular pathogen, the infection involves macrophages, monocytes of peripheral blood, macrophages of peripheral tissues, premonocytes and monocytes of bone marrow.
In immunocompetent patients the interaction between mycobacterium- macrophage activates the macrophage and as a consequence bacterial lysis due to enzymatic digestion and oxidation. On the contrary, in immunocompromised patients, the inability of a bactericidal action is considered the main cause of the systemic spread of the mycobacterium.
In merit to the pathogenicity mechanisms and the virulence factors, molecular data reports an involvement of elements in the structural characterization of bacterial walls of the micro-organism (lipoarabinomannan, sulfatide, and proteins) in particular for the activation of cell-mediated immunitary response.
The sulfatide seems to be the first responsible for macrophage activation and for the increased secretion of IL-1β and TNF-α, favouring the formation of granulomas that slowly develop causing extended tissue destruction.
The observed histopathological lesions can be exudative lesions, characterized by acute inflammation, exudate formation, and accumulation of PMN leucocytes around the bacilli, and productive lesions (granulomatous) characterized by concentric distribution of macrophages (epithelioid cells) to form tubercles and giant cells.
Even though the initial diagnosis of Mycobacteria infection is often based on clinical data, the definitive diagnosis requires isolation and identification of the micro-organism in a laboratory.
Classic laboratory procedures for the analysis of clinical samples involve the decontamination and digestion of the specimen, the direct microscopic detection of the presence of AFB (acid – fast bacilli) after staining of the slides prepared with infectious materials, with Ziehl-Neelsen, the staining with rhodamine-auramine fluorescent mix, the isolation of the micro-organism in culture, and test of drug resistance.
Due to the reduced growing speed of Mycobacteria, their isolation requires some weeks (at least 3-9 weeks with optimum temperature at 37°C).
In the last ten years different molecular methods for direct identification and typing of Mycobacteria have been developed.
Mycobacterium tubercolosis typing by means of the classical methods based on the phenotype, on the biochemical characteristics, and on the use of chromatographic techniques, requires a long execution time, not suitable for routine screening. PCR-based methods produce the same information with less time and costs (Stauffer et al., 1998; Glennon et al., 1994).
Genotyping is based on the presence of specific sequences on precise genes (katG-463, gyrA-95) and on their association with insertion sequences (IS6110) (Telenti et al., 1993; Fries et al., 1990; Hance et al., 1989).
9. TEST PRENSİBİ
PCR metodu (Polimeraz Zincir Reaksiyonu) literatürde DNA amplifikasyonu için tanımlanan ilk metoddur (Saiki RK ve arkadaşları, 1985). Thermostable DNA polymerase ile DNA nın spesifik bir parçasının (hedef dizi) in vitro amplifikasyon reaksiyonu olarak tanımlanabilir.
Reaksiyonda üç nükleik asit segmenti bulunur: Amplifiye olması için çift iplikli DNA kalıbı (hedef DNA) ve iki tek iplikli oligonükleotid “primerler” kalıp DNA ya spesifik olarak dizayn edilmiştir.
DNA polymerase primerler tarafından işaretlenen bölgeden sentez işlemine başlar ve solusyonda serbest olarak bulunan tamamlayıcı nükleotidlerin (dNTPler) eklenmesi ve bağlanması ile orjinal çift iplikli hedef DNA bölgesi ile aynı olan yeni çift iplikli DNA molekülünü sentezler. Çeşitli sikluslardan sonra, hedef diziye uygun olan milyonlarca DNA molekülü oluşturabilir.
Bu testin duyarlılığı, laboratuar tanısında uygulamaya olanak sağlar.
Moreover, the amplification reaction can be executed from a wide range of biological samples, and since, this technique is able to amplify very small DNA segments, the starting DNA can be even partially degraded.
10. ÜRÜN TANIMI
The method of MYC-TE kit consists of the amplification of a highly conserved region (hsp65, heat shock protein, 65 kDa membrane protein) of Mycobacterium tuberculosis genome, allowing the identification of a high number of Mycobacterium species.
Successively, the amplified product can be typed by digestion with restriction enzymes (RFLP) that cut the double-stranded DNA in specific sites of known sequences.
As the 65 kDa protein amplified sequence changes in function of the Mycobacterium type, the digestion leads to a restriction profile which allows the identification of Mycobacterium species.
Using this method, it is possible to detect about 40 different Mycobacteria, including all the most important Mycobacteria with clinical relevance. This region is highly variable (in the nucleotide composition), also interspecies require clone distinctions for certain types of Mycobacteria.
The kit can also evaluate the suitability of the extracted DNA for the amplification, by amplification of the TST- thiosulfate sulfurtransferase (rhodanese) gene in the 22q13.1 region (amplification control). A negative result in the amplification of the TST- thiosulfate sulfurtransferase
(rhodanese) gene indicates either the presence of amplification reaction inhibitors or that the DNA is highly degraded. This method helps the operator to recognize possible false negative results for the Mycobacterium, and no extra time is needed, so that the amplification of the TST gene can be done contemporarily with the amplification of the Mycobacterium.
The kit also provides the positive control for the amplification process. When the amplification of the positive control is successful, it guarantees that the reaction was performed properly. These controls are not dangerous for the operator, because they are plasmid DNA, containing only a part of Mycobacterium genome.
The kit is in a premix format: all the reagents for the amplification are premixed and aliquoted in single dose test-tubes (0,2 or 0,5 mL), which will be added with Taq polymerase and extracted DNA.
This premix format reduces the number of steps in pre-amplification, with considerable time saving for the operator. Above all, this format minimizes the risk of contamination, so the risk to get false positive results is significantly reduces. Avoid repeated freezing/thawing of reagents (that could alter the product performances).
In any case, it is always recommended to use all the proper amplification controls.
11. ÖRNEK TOPLAMA, MANİPULASYON VE ÖN- MUAMELESİ
The samples used for the determination of Mycobacterium infection by microbiological analysis and molecular biology are respiratory specimens (saliva, sputum, expectorates, bronchial secretions obtained by bronchoscopy, bronchoalveolar washings) and other kinds (pleuric essudates, pulmonary, lymphonodal and cutaneous and pleuric biopsies, peripherial blood, gastric aspirates that contain swallowed excretions, cerebral fluid, and bone marrow), as well as cultures (Riggio et al., 1997;
11.1. Respiratory specimens
During the collection of biological samples, it is a good safety rule to consider every sample as potentially infectious and handle it as such.
Expectorate collection and bronchoscopy, which could cause diffusion in the environment of infected mycobacteria, should be done in suitable rooms and the staff should use appropriate protection barriers.
Respiratory samples can be liquified by adding N-acetil-L-cysteine (1,5%) or dithiothreitol (0,1%), in equal volume with the sample (directly on the collection box) and left at 37°C for 15-30 min according to the material’s consistency.
Generally, respiratory samples are decontaminated by adding 4% NaOH solution, then, after centrifugation, neutralized with HCl. The obtained pellet can be resuspended in PBS.
Collection boxes should be sterile, disposable, and with screw cap. To have a significant analysis, the sample should not be saliva, but a sample from the deep respiratory tract.
Fresh or decontaminated samples should be stored at +2 / +8°C for a short time (no more than 48 hours), or at -20°C for a longer period (also for some months).
11.2. Histological samples
Histological samples are pulmonary, skin, lymphonodal, and pleural biopsies, fresh, frozen, formalin-fixed or paraffin embedded biopsies.
The biopsy sample collection should be performed as routine.
Fresh biopsies can be used within a few minutes from sampling, or quickly frozen with liquid nitrogen and successively stored at -80°C until mechanically disintegrated by using a sterile cutter, followed by enzymatic digestion.
In case, the biopsy is fixed and paraffine-embedded, it is suggested to use formalin buffered at pH 7 with sodium and potassium salts at 10%, as in a Lilie formula. Tissue fixation with unbuffered formalin in Bouin, Holland, or other acidic fixatives (osmic acid, for instance) are not suitable for subsequent DNA extraction, because these substances produce cross-links in the tissue, making it indigestible.
In case of fresh or frozen histological samples (until about 50 mg), it is suggested to proceed immediately with the mechanic disintegration of the tissue by using a sterile razorblade. Do this operation on a glass slide, on which an aliquote of 1X PBS buffer is added. Then transfer the minced tissue in a tube by a Pasteur pipette, and proceed with the sample’s digestion.
If the histological sample is fixed and paraffin embedded, proceed with the paraffin removal and then with the sample’s enzymatic digestion.
11.3. Blood
Mycobacterium detection may be done on peripheral blood, which can be the best medium in case of extrapulmonary or scattered tuberculosis.
The sample collection should follow all the routine sterility precautions, and transported in sterile boxes, without transport medium.
Kan EDTAlı olarak alınmalıdır. Heparin gibi diğer koagülasyon ajanları TAQ polymerase’ın güçlü inhibitörleridir ve bu yüzden amplifikasyon reaksiyonunun verimliliğini bozar.
Fresh blood can be stored at +2 / +8°C (processed within 4 hours from the collection); if DNA is not extracted immediately, it is necessary to freeze the sample at -20°C.
It is necessary to do another step by Ficoll-Hypaque system or erythrocyte lysis protocols which allows the isolation of lymphocytes.
11.4. Cerebral fluid
Also for this clinical material, sample collection should follow all the routine sterility precautions, and the sample should be transported in sterile boxes, with transport buffer (1X PBS).
In this case, decontamination is not necessary. Collect the maximum possible volume.
Fresh cerebral fluid can be immediately processed after the collection, or can be stored at +2 / +8°C (if processed within 4 hours from the collection);
freezing the sample is not suggested.
12. SCREENING PROTOCOL
12.1. DNA extraction
For DNA extraction, any DNA extraction method can be used, provided that it extracts pure and integral DNA.
In particular, some good results have been obtained with an extraction system which causes cellular lysis by repeated steps of freezing/boiling.
During these steps, it could be useful to add Tris-EDTA and Chelex 100 to the sample; these solutions stabilize the DNA during the boiling step, maintaining the ionic force of the sample.
In literature, the combination of the first step of freezing/boiling with bacteria lysis by Lisozyme and Proteinase K digestion (Niyaz et al., 1998) is described.
In any case, it is necessary to remember that the extraction volumes indicated in paragraphs 11.2.1 and 11.3.1 for TST and Mycobacterium amplification (20 μL) are referred to extracts obtained with AB ANALTICA methods.
When using an alternative extraction method, in case the volume of the DNA sample for amplification is less than 20 μL, it will be necessary to adjust the mix amplification volume by adding water.
12.2. DNA amplification
12.2.1. TST DNA amplification
Add to each blue premixed test-tube:
AB Taq 0.5 μL
extracted DNA 20 μL
It is important to include in each experiment a negative control to monitor the contamination (add distilled water to the mix instead of extracted DNA) and a positive control (any genomic DNA).
12.2.2. Amplification of Mycobacterium DNA
Add to each colourless premixed test-tube:
AB Taq 0.5 μL
Extracted DNA 20 μL
Always amplify, with the sample to be analyzed, a negative control (add distilled water to the mix instead of DNA) and a positive control (20 µL of the positive control included in the kit).
Centrifuge briefly and put the test-tubes (TST and Mycobacterium) in the thermalcycler programmed as below:
1 cycle 94°C 3 min 40 cycles
95°C 60°C 72°C
60 sec 60 sec 60 sec
12.3. Amplifikasyon ürünlerinin görüntülenmesi:
12.3.1. Agaroz jel elektroforezi
For visualization of amplification product prepare a 3% agarose, weighting 1,5 g of agarose and add 50 mL of 1X TAE to it.
Solusyonu berrak oluncaya kadar manyetik ısıtıcı ya da mikrodalga içine bırakın. Jeli el ile dokunabilecek kadar soğumaya bırakın ve 10 µL Ethidium Bromide (2,5mg/mL) ekleyin.
NOT: Ethidium Bromide güçlü bir mutajenik ajandır: Daima eldiven kullanın ve bu reaktifle ya da içeren jel ile çalışırken tercihen kimyasal güvenlik kabini altında çalışın.
Jeli tarakları ile birlikte uygun bir dökme tankına koyun ve oda ısısında soğuyana kadar bekleyin ya da jel katı hale gelene kadar buzdolabına koyun.
Jel katılaştığında, dikkatlice tarakları çıkarın (jel kuyucuklarına zarar vermemeye dikkat edin), tankı elektroforez haznesine alın ve jelin üzerini tamamen kaplayana kadar uygun miktarda TAE buffer ekleyin (yaklaşık jel yüzeyinin 1-2mm üzerinde).
12.3.2. Örnek yükleme
Test tüpünde ya da direk parafilmde aşağıdakileri karıştırın:
2 µL 6X Blue
10 µL PCR products or DNA molecular weight marker (MW Marker*)
Load the mixture in the gel’s wells; switch on the power supply and set the voltage between 80-100 V. At the end of the run, place the gel on an UV transilluminator and analyze the results by comparing the size of the amplification products with the reference DNA Molecular Weight Marker.
* = 6X Blue and MW Marker PM are included in the 03-18A kit; for another loading buffer or molecular weight marker follow the supplier’s instructions.
DNA Moleküler Ağırlık Markır (MW)
501- 489, 404, 331, 242, 190, 147, 111, 110, 67, 34x2, 26 bp.
%2 agaroz jelde 501-489 bp bantlar genellikle tam olarak çözülmez ve tek bir bant olarak görülür; 26 ve 34 bp bantları bazen görünmek için çok küçük olur (düşük moleküler ağırlıklarından dolayı).
NOT: UV ışınları deri ve en önemlisi gözler için tehlikelidir: daima eldiven ve koruyucu gözlük kullanın ya da UV transilluminatorün koruyucu ekranını kullanın.
12.3.3. Sonuçların yorumlanması TST and Mycobacterium DNA amplification
The included controls should show the following results:
CONTROL RESULT INTERPRETATION
Positive control Positive The PCR amplification functions properly
Negative control Negative Absence of contaminates
The interpretation of the bands on agarose gel is as follows on the table below:
RESULTS INTERPRETATION TST band is absent
Sample not suitable for amplification (repeat the DNA extraction)
TST band is present, but the Mycobacterium band is absent
Amplifiable sample
Sample is Mycobacterium negative
13. TYPING PROTOCOL
13.1. Typing by digestion with restriction enzymes (RLFP)
For each positive sample, mix into two different test tubes for enzyme 1 and enzyme 2:
Add two drops of mineral oil, then centrifuge briefly and incubate one hour at 37°C in the thermalcycler.
(If the amplified product band is too weak, it is suggested to digest more DNA and to add less water, so the final volume of 30 µL is maintained).
13.2. Amplifikasyon ürünlerinin görüntülenmesi:
13.2.1. High resolution agarose gel electrophoresis (HR)
For visualization of the restriction bands, prepare a 3% HR agarose gel.
Follow the instructions at paragraph. 12.3.1.
13.2.2. Örnek yükleme
Test tüpünde ya da direk parafilmde aşağıdakileri karıştırın:
3 µL 6X Blue*
15 µL Digested amplified product
or DNA molecular weight marker (MW Marker)
Load the mixture on gel wells; switch on the power supply and set the voltage between 80-100 V. At the running end, place the gel on an UV transilluminator and analyze the results by comparing the size of the amplification products with the reference DNA Molecular Weight Marker.
Sterile water 10.5 µL 10X Enzyme 2 Buffer 3 µL
Enzyme 2 0.5 µL
Amplified DNA 15 µL Mineral oil 2 drops Sterile water 10.5 µL
10X Enzyme 1 Buffer 3 µL
Enzyme 1 1.5 µL
Amplified DNA 15 µL Mineral oil 2 drops
Molecular Weight Marker (MW Marker in 03-18A):
501- 489, 404, 331, 242, 190, 147, 111, 110, 67, 34-{}-x2, 26 bp.
%3 agaroz jelde 501-489 bp bantlar genellikle tam olarak çözülmez ve tek bir bant olarak görülür; 26 ve 34 bp bantları bazen görünmek için çok küçük olur (düşük moleküler ağırlıklarından dolayı).
NOT: UV ışınları deri ve en önemlisi gözler için tehlikelidir: daima eldiven ve koruyucu gözlük kullanın ya da UV transilluminatorün koruyucu ekranını kullanın.
13.2.3. Sonuçların yorumlanması
For interpretation of the restriction results consult Table 1 on the next page, which reports the restriction profiles of the recognised species of Mycobacterium.
Table 1
Enzyme 1 (bp) Enzyme 2 (bp) SPECIES No digestion 180 / 140 M. trivale No digestion 175 / 80 / 65 M. vaccae No digestion 140 / 105 / 70 M. szulgai
No digestion 140 M. flavescens
325 140 210 M. chelonae
325 140 175 / 115 M. haemophilum
325 120 190 / 140 M. terrae
325 120 180 / 150 M. aurum
325 120 155 M. nonchromogenicum II
325 120 140 / 115 / 70 M. gordonae IV
325 120 140 / 105 M. genevense
325 120 140 / 65 / 60 presumtive M. chelonae 245 220 200 / 135 M. simiae I
245 220 160 / 115 / 80 M. marinium
245 220 160 / 60 M. abscessus
245 220 155 / 150 / 100 M. peregrinum 245 220 155 / 140 M. simiae II 245 220 155 / 135 / 95 M. scrofulaceum 245 220 150 / 100 M. flavescens II 245 220 140 / 105 / 80 M. kansasii
245 220 140 / 105 M. avium
245 220 115 / 110 M. asiaticum 245 140 / 80 175 / 80 M. aurum 245 140 / 80 160 / 135 / 60 M. smegmatis 245 140 / 80 150 / 110 / 70 M. shimoidei 245 140 / 80 140 / 120 / 95 M. gordonae VI
245 140 / 80 140 / 105 / 70 M. gasatri / M. Kansasii 245 120 / 100 155 / 140 / 60 M. intracellulare/MAI St.18 245 120 / 100 155 / 110 / 70 M. malmoense
245 120 / 100 140 / 120 M. gordonae III 245 120 / 80 235 / 115 M. gordonae II 245 120 / 80 170 / 115 / 60 M. gordonae I 245 120 / 80 170 / 105 M. xenopi
245 120 / 80 160 / 140 / 70 M. tuberculosis complex 245 120 / 80 160 M. nonchromogenicum I 245 120 / 80 155 / 135 M. fortuitum
It is possible that samples that are positive at the screening step, when subjected to restriction analysis, give undigested products or very complex restriction patterns; this can depend on the type of the clinical sample, in particular with paraffin-embedded specimens.
For any further information contact AB ANALITICA technical support e-mail:
laboratorio@abanalitica.it, fax +39 (049)-761698, or tel. +39 (049)-8709510.
Figure 1: 3% HR agarose gel electrophoresis 1: DNA Molecular Weight marker
2: Sample A restriction by enzyme 1
14. SORUN GİDERME
Hem amplifikasyon ürünü hem de pozitif control DNA bandı yoksa
• TAQ polymerase premikse doğru eklenememiştir
- -Uygun volümde pipet ve uçlar kullanın (pipet aralığı 0,2-2 μL);
- -Görsel olarak TAQ polymerasın premikse difüze olduğunu gözleyin:
Bu basittir, çünkü enzim, yüksek bir densiteye sahip olan gliserolde çözülür;
- Alternative olarak, tüp duvarına konulmuş olan TAQ polymerasın görsel olarak teyit edin, sonra kısa bir süre santrifüj edin.
• Thermalcycler doğru olarak programlanmamıştır
- Check that the thermalcycler program conforms with the temperature profile in the instruction manual. Repeat the DNA amplification with the correct program if the thermalcycler was programmed incorrectly.
• Kit uygun şekilde çalışmaz
- -Premiks, TAQ polymerase ve pozitif kontrolü -20°C de saklayın;
- -Premiks ve reaktifleri tekrar tekrar dondurup/çözdürmekten sakının.
No amplification bands for TST or Mycobacterium in the tested sample, but a good band for positive controls
• Ekstraksiyon aşaması sırasında olası problemler:
- Be sure that you follow correctly all the instructions for the extraction kit used;
- Consult the troubleshooting section of the extraction kit used;
- Yeni bir örnek ile doğru şekilde tekrar DNA ekstraksiyonu yapın.
• The amplification was inhibited
- Dilute the starting sample with distilled water and TE;
- Repeat the DNA extraction from a smaller amount of clinical sample;
- Use an adequate extraction system.
Presence of aspecific products or extra bands after the visualization of the amplified product in agarose gel
• The thermalcycler makes temperature changes too slowly - Execute a thermalcycler revision.
• The preparation of the amplification reaction was done for a long time at room temperature.
- Accelerate the work time at room temperature;
- Work on ice.
• The extracted product was not purified
- Use an extraction system which purifies adequately.
• The starting sample contained degraded DNA
- Repeat the extraction step using another starting clinical sample;
- Be sure that the sample was collected and stored appropriately.
For any further problems contact AB ANALITICA’s technical support: e-mail:
laboratorio@abanalitica.it; fax +39 (049)-8709510, or tel. +39 (049)-761698
15. TEST LİMİTLERİ
Kit performansı aşağıdaki durumlarda azalır:
• The clinical sample is not suitable for this analysis (use of alternative fixatives instead of neutral formalin buffer for histological samples or incorrect sample storing).
• The DNA is not amplifiable because of the presence of amplification reaction inhibitors or due to an inadequate extraction system.
• The kit was not stored at the suggested temperatures.
16. TEST PERFORMANSI
16.1. Özgünlük
En önemli bilgi bankasındaki primer dizi sıralaması spesifik olmayan dizilerin yokluğunu gösterir ve farklı STR ve VNTR markırlarının spesifik amplifikasyonunu garanti eder.
Cross reactions with genomic DNA or other pathogenic microorganism nucleic acid have not been revealed.
16.2. Diagnostic significance
Generally, the diagnosis of tubercular disease can be clinically significant even with the isolation of only one bacillus; the infection of non tubercular mycobacteria (except for the cases of isolation of pathogen species as M.
avium/intracellulare, M. kansasii e M. scrofulaceum), there are different protocols based on diagnostic criterions. Many authors agree that it is necessary to check at least one of the following conditions: repeated isolation, presence of high microbial charges, and presence of clinical symptomatology (Tsukamura, 1986).
16.3. Diagnostic and analytic sensitivity
Some bibliographic references have demonstrated that PCR is a very sensitive system able to find 10 tubercular bacillus in a cell population of 106. Its diagnostic sensitivity is about 84%.
17. REFERANSLAR
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Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA and Arnheim N, Science 230, 1350-1354, 1985
Pai S, Esen N, Pan X, Musser JM. Arch Pathol Lab Med 121, 859-864, 1997.
Riggio MP, Gibson J, Lennon A, Wray D, McDonald DG. Gut 41, 646- 650,1997.
Gulletta E. Microbiol Med, 10 (2), 41-44, 1995.
Niyaz A, Mohanty AK, Mukhopadhyay U, Batish VK, Grover S. J Clin Microbiol 36 (10), 3094-3095, 1998.
Wang S X e Tay L. Jour of Clin Microb, 1932-1934, June 1999.
Folgueira L, Delgado R, Palenque E, Aguado J M, Noriega A R. Jour of Clin Microb, 512-515, Mar 1996.
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