PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR)
Contamination of pathogenic bacteria Staphylococcus aureus in milk samples have been analyzed using Real Time Polymerase Chain Reaction (RTi- PCR) with target of nuc gene. The research aims to develop rapid analysis method S. aureus contamination in milk. The study was divided into three stages whic...
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| Format: | Thesis |
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[Yogyakarta] : Universitas Gadjah Mada
2014
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| author | , RISNITA VICKY L , Dr. Tri Joko Raharjo, M.Si. |
| author_facet | , RISNITA VICKY L , Dr. Tri Joko Raharjo, M.Si. |
| author_sort | , RISNITA VICKY L |
| collection | UGM |
| description | Contamination of pathogenic bacteria Staphylococcus aureus in milk
samples have been analyzed using Real Time Polymerase Chain Reaction (RTi-
PCR) with target of nuc gene. The research aims to develop rapid analysis method
S. aureus contamination in milk.
The study was divided into three stages which are development, validation
and application of the method. Method development comprised isolation of S.
aureus DNA, quantification of DNA by spectrophotometry UV-VIS and
amplification by RTi-PCR. Amplification was performed by testing annealing
temperature (Ta) at 58 � 60 °C for obtaining optimal Ta. RTi-PCR condition are
predenaturation at 95°C for 3 minutes, denaturation at 95 °C for 5 seconds,
annealing at Ta optimal for 30 seconds with Melting Curve Analysis. Method
validation consisted of spesificity test, precision test and cut off detection test.
Spesificity of the method was checked using artificially S. aureus contaminated
milk with uncontaminated sample. Precision was tested by performing repeated
analysis artificially S. aureus contaminated milk. Cut off detection was tested by
analyzing contaminated milk with variation of the bacteria concentration.
Application method was performed by testing of commercial milk.
The result show RTi-PCR method with primer nuc A1 and nuc A2 is
spesific for DNA detection of S. aureus with Tm (melting temperature) amplicon
79 °C. Optimal Ta for PCR is 58 °C. The method show high spesificity for
detecting artificially S. aureus contaminated milk. Repitability test show
artificially S. aureus contaminated milk samples have Tm amplicon 78,5 � 79 °C
and average Ct (Cycle number threshold) 32,63 ± 1,08. RTi-PCR method can
detect S. aureus up to 1,04 � 101 CFU/mL in the sample. RTi-PCR analysis for
commercial milk show two samples of pasteurized milk, one milk sample sold in
traditional market and one milk sample sold in a small cafe are contaminated with
S. aureus whereas UHT milk sample is not. |
| first_indexed | 2024-03-13T23:16:13Z |
| format | Thesis |
| id | oai:generic.eprints.org:127162 |
| institution | Universiti Gadjah Mada |
| last_indexed | 2024-03-13T23:16:13Z |
| publishDate | 2014 |
| publisher | [Yogyakarta] : Universitas Gadjah Mada |
| record_format | dspace |
| spelling | oai:generic.eprints.org:1271622016-03-04T08:09:50Z https://repository.ugm.ac.id/127162/ PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR) , RISNITA VICKY L , Dr. Tri Joko Raharjo, M.Si. ETD Contamination of pathogenic bacteria Staphylococcus aureus in milk samples have been analyzed using Real Time Polymerase Chain Reaction (RTi- PCR) with target of nuc gene. The research aims to develop rapid analysis method S. aureus contamination in milk. The study was divided into three stages which are development, validation and application of the method. Method development comprised isolation of S. aureus DNA, quantification of DNA by spectrophotometry UV-VIS and amplification by RTi-PCR. Amplification was performed by testing annealing temperature (Ta) at 58 � 60 °C for obtaining optimal Ta. RTi-PCR condition are predenaturation at 95°C for 3 minutes, denaturation at 95 °C for 5 seconds, annealing at Ta optimal for 30 seconds with Melting Curve Analysis. Method validation consisted of spesificity test, precision test and cut off detection test. Spesificity of the method was checked using artificially S. aureus contaminated milk with uncontaminated sample. Precision was tested by performing repeated analysis artificially S. aureus contaminated milk. Cut off detection was tested by analyzing contaminated milk with variation of the bacteria concentration. Application method was performed by testing of commercial milk. The result show RTi-PCR method with primer nuc A1 and nuc A2 is spesific for DNA detection of S. aureus with Tm (melting temperature) amplicon 79 °C. Optimal Ta for PCR is 58 °C. The method show high spesificity for detecting artificially S. aureus contaminated milk. Repitability test show artificially S. aureus contaminated milk samples have Tm amplicon 78,5 � 79 °C and average Ct (Cycle number threshold) 32,63 ± 1,08. RTi-PCR method can detect S. aureus up to 1,04 � 101 CFU/mL in the sample. RTi-PCR analysis for commercial milk show two samples of pasteurized milk, one milk sample sold in traditional market and one milk sample sold in a small cafe are contaminated with S. aureus whereas UHT milk sample is not. [Yogyakarta] : Universitas Gadjah Mada 2014 Thesis NonPeerReviewed , RISNITA VICKY L and , Dr. Tri Joko Raharjo, M.Si. (2014) PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR). UNSPECIFIED thesis, UNSPECIFIED. http://etd.ugm.ac.id/index.php?mod=penelitian_detail&sub=PenelitianDetail&act=view&typ=html&buku_id=67406 |
| spellingShingle | ETD , RISNITA VICKY L , Dr. Tri Joko Raharjo, M.Si. PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR) |
| title | PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR) |
| title_full | PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR) |
| title_fullStr | PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR) |
| title_full_unstemmed | PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR) |
| title_short | PENGEMBANGAN METODE ANALISIS CEPAT CEMARAN BAKTERI PATOGEN Staphylococcus aureus DALAM SUSU DENGAN REAL TIME POLYMERASE CHAIN REACTION (RTi-PCR) |
| title_sort | pengembangan metode analisis cepat cemaran bakteri patogen staphylococcus aureus dalam susu dengan real time polymerase chain reaction rti pcr |
| topic | ETD |
| work_keys_str_mv | AT risnitavickyl pengembanganmetodeanalisiscepatcemaranbakteripatogenstaphylococcusaureusdalamsusudenganrealtimepolymerasechainreactionrtipcr AT drtrijokoraharjomsi pengembanganmetodeanalisiscepatcemaranbakteripatogenstaphylococcusaureusdalamsusudenganrealtimepolymerasechainreactionrtipcr |