Interpretation of the Result of Biochemical Test gdna extraction Gel electrophoresis

Transcription

1 Interpretation of the Result of Biochemical Test gdna extraction Gel electrophoresis Kamolchanok Rukseree Phajongjit Karraphan Laboratory Training and Risk Management Workshop, Reducing Biosecurity Threats from Infectious Diseases with Pandemic Potential in Southeast Asia, Mahidol University Amnatcharoen Campus, 9 July 2015

2 Guidelines for Environmental Sampling of Burkholderia pseudomallei 1. Morphology 2. Latex Agglutination Test 3. Antimicrobial Susceptibility Test 4. L-arabinose agar, Glucose agar & Ashdown agar Note: No need to do molecular diagnosis Limmathurotsakul D, et al. Farrar J, ed. PLoS Neglected Tropical Diseases. 2013;7(3):e2105. doi: /journal.pntd

3

4 Biochemical Test Biochemical test Triple Sugar Iron (TSI) Agar Slant = Slant: variable; Butt = red (no change), TSI = No change (N/N) TSI

5 Oxidase Test Oxidase : Positive

6 L-arabinose and glucose assimilation NSS BP on LB agar 0.5 MacFaland Drop 3 ul L-Arabinose agar Drop 3 ul Incubate at 37 0 C, 48 hrs Glucose agar The glucose agar was used as control for all isolates

7 L-arabinose and glucose assimilation Growth No Growth Glucose assimilation B. pseudomallei Non L-arabinose assimilation

8 Charateristics of B. pseudomallei TSI : No change (N/N) Oxidase : Positive Latex agglutination : Positive Susceptibility to amoxicillin-clavulanate (clear zone around) Non L-arabinose assimilation

9 Biochemical Test using API 20 NE The analytical profile index or API is a classification of bacteria based on experiments and is developed for quick identification of clinically relevant bacteria. Because of this, only known bacteria can be identified. API test strips consists of microtubes (cupules) containing dehydrated substrates to detect the enzymatic activity or the assimilation / fermentation of sugars by the inoculated organisms. During incubation, metabolism produces colour changes that are either spontaneous or revealed by the addition of reagents. When the carbohydrates are fermented, the ph within the cupule changes and is shown by an indicator. Assimilation tests are inoculated with a minimal medium (API AUX medium) and the bacteria grow if they are able to utilize the corresponding substrate: a positive result is indicated by growth. Test results are entered into an online database to determine the bacterial identity.

10 API 20 NE kit API 20 NE 8 Conventional test 12 Assimilation test

11 Reading table

12 Interpretation 24 hrs. 48 hrs Burkholderia pseudomallei

13 BP_Ref 01 (24 hrs.) Before adding reagents After adding reagents

14 T4L/A3, St.

15 Results T2L/C1,St. T3L/B3,St.

16 Results T4L/B2, Sp. 1 T4L/B3, St

17 Results Isolate TSI (N/N) Oxidase Latex agglutination AST (cm.) L-Arabinose Glucose API20NE profile 1 T2R/C3,Sp N/N + + S (3.45) NG (Ara-) G T2L/C1,St N/N + + S (3.35) NG (Ara-) G T3L/B3,St N/N + + S (3.23) NG (Ara-) G T3L/B3,Sp N/N + + S (3.30) NG (Ara-) G T4L/A1,Sp N/N + + S (3.23) NG (Ara-) G T4L/A2,St (LA +) N/N + + S (3.20) NG (Ara-) G T4L/A2,Sp N/N + + S (3.30) NG (Ara-) G ND 8 T4L/A2 N/N + + S (2.90) NG (Ara-) G ND 9 T4L/A3,St N/N + + S (3.40) NG (Ara-) G T4L/B1,St 2 N/N + + S (3.45) NG (Ara-) G T4L/B2,Sp 1 N/N + + S (3.35) NG (Ara-) G T4L/B2,Sp 2 N/N + + S (3.50) NG (Ara-) G T4L/B3,St N/N + + S (3.50) NG (Ara-) G T4L/B3,Sp N/N + + S (3.30) NG (Ara-) G ND 15 T4L/C1,St N/N + + S (3.30) NG (Ara-) G T4L/C1,Sp N/N + + S (3.45) NG (Ara-) G ND 17 T4L/C2,St (Type V) N/N + + S (3.30) NG (Ara-) G T4L/C2,Sp N/N + + S (3.50) NG (Ara-) G T4L/C3,St N/N + + S (3.55) NG (Ara-) G T4L/C3,Sp N/N + + S (3.30) NG (Ara-) G ND 21 T4L(Mix),St 1 N/N + + S (3.40) NG (Ara-) G T4L(Mix),St 2 N/N + + S (3.50) NG (Ara-) G T4L(Mix),St 3 N/N + + S (3.70) NG (Ara-) G T4L(Mix),Sp N/N + + S (3.50) NG (Ara-) G ND = Not determine, NG = No growth, G = Growth

18 Conclusions Numerical profile BP = , , BT = , , We found that numerical profile is Mostly, B. pseudomallei were found at the left side of the main road.

19 Genomic DNA isolation Genomic DNA is extracted from the bacteria using the Mini gdna Bacteria Kit. Lane 1 = 1KB plus marker Lane 2 = 1st Bp_Ref.01 elute 100 ul Lane 3 = 2nd Bp_Ref.01 elute 100 ul Lane 4 = 1st T2R/C3 elute 50 ul Lane 5 = 2nd T2R/C3 elute 50 u Lane 6 = 1st T3L/B3 elute 100 u Lane 7 = 2nd T3L/B3 elute 100 u Lane 8 = 1st T1L/C3 elute 200 u Lane 9 = 2nd T1L/C3 elute 200 u

20 Genomic DNA isolation 1 1. Streak the bacteria on LB agar plate and incubate at 37 C for 16 hours 2. Add 500 µl of phosphate buffer saline (PBS) to a 1.5ml microcentrifuge tube. 3. Re-suspend a loopful of the bacteria in PBS thoroughly. 4. Centrifuge at 14,000 16,000 g for 1 minutes to pellet the cells. Remove the supernatant. 5. Add 180 µl of GT Buffer then re-suspend the cell pellet by vortex or pipette. Add 20 µl of Proteinase K (make sure ddh 2 O is added). Incubate at 60ºC for at least 10 minutes. During incubation, invert the tube every 3 minutes. 6. Add 200 µl of GB Buffer to the sample and mix by vortex for 10 seconds. Incubate at 70ºC for at least 10 minutes to ensure the sample lysate is clear. During incubation, invert the tube every 3 minutes.

21 Genomic DNA isolation 2 7. Add 200 µl of absolute ethanol to the sample lysate and mix IMMEDIATELY by shaking vigorously. If precipitate appears, break it up as much as possible with a pipette. Place a GD Column in a 2 ml Collection Tube. Transfer mixture to the GD Column then centrifuge at 14-16,000 x g for 2 min. Discard the 2 ml Collection Tube containing the flow-through then place the GD Column in a new 2 ml Collection Tube. 8. Add 400 µl of W1 Buffer to the GD Column. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Place the GD Column back in the 2 ml Collection Tube. Add 600 µl of Wash Buffer (make sure ethanol was added) to the GD Column. Centrifuge at 14-16,000 x g for 30 s then discard the flow-through. Place the GD Column back in the 2 ml Collection Tube. Centrifuge again for 3 m at 14-16,000 x g to dry the column matrix. 9. Transfer the dried GD Column to a clean 1.5 ml microcentrifuge tube. Add 100 μl of pre-heated Elution Buffer 1, TE Buffer 2 or water 3 into the CENTER of the column matrix. Let stand for at least 3 minutes to allow Elution Buffer, TE Buffer or water to be completely absorbed. Centrifuge at 14-16,000 x g for 30 s to elute the purified DNA.

22 PCR amplification PCR amplification (20 μl) is carried out on chromosomal DNA using Taq polymerase with an initial denaturation time of 4 min at 95 o C, followed by 30 cycles of 95 o C for 30 s, 62 o C for 30 s and 72 o C for 60 s. Samples are then maintained at 72 o C for a further 10 min, cooled to 4 o C and stored at -20 o C. 1% agarose gel, 100 volt, 40 minute gltb gene Lane 1 = 1KB plus marker Lane 2 = T2R/C3, Sp Lane 3 = T2L/C1, St Lane 4 = T3L/B3, St Lane 5 = T3L/B3, Sp GoTag Green Master Mix