Mutation Detection by Fluorescent Hybridization Probe Melting Curves

Bernard, Philip S.; Reiser, Astrid; Pritham, Gregory H.

doi:10.1007/978-3-642-59524-0_2

Cited by 12 publications

(11 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is known that the shifts gre a ter than 1 o C from the cha rac te ris tic (ho mo dup lex in this si tu a ti on) de riva ti ve mel ting cur ve pro fi le arises a sus pi ci on of mu ta ti on. 29 By re al-ti me as say the elec trop ho re sis step is skip ped, thus the the time needed for the met hod is shor te ned re mar kably. Re al-ti me he tero dup lex analy sis may be a ra pid and cost-ef fec ti ve al ter na ti ve to ot her mo le cu lar met hods for se rotype pre dic ti on of pne u mo coc ci.…”

Section: Discussionmentioning

confidence: 99%

Serotype Prediction for Frequently Isolated Serotypes of Streptococcus Pneumoniae by Heteroduplex Analysis and Modification of This Technique to Real-Time Fluorometric Nucleic Acid Detection System

Pinar¹,

Şener²,

Ustaçelebi³

2010

Turkiye Klinikleri J Med Sci

View full text Add to dashboard Cite

A AB BS S T TR RA AC CT T O Ob b j je ec c t ti i v ve e:: Se ve ral mo le cu lar se roty pe pre dic ti on met hods ha ve be en pub lis hed in or der to simp lify the pne u mo coc cal se roty ping. The aim of this study is to de ve lop a mo le cu lar se roty pe pre dic ti on tech ni qu e al ter na tive to ot her mo le cu lar ap pro ac hes. M Ma a t te e r ri i a al l a an nd d M Me et t h ho od ds s: : S. pne u mo ni a e se roty pes 1, 3, 5, 6A, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F and 23B, that are the most fre qu ently iso la ted se roty pes from in fec ti ons in Tur key, we re inc luded in the study. S. pne u mo ni a e cpsA and cpsB ge nes that are in vol ved in the pro ces sing, re gu la ti on and ex port of the cap su lar poly sacc ha ri des, we re amp li fi ed by polymerase chain reacsion (PCR). Then, he te ro dup lex analy sis was per for med to the 1.8 kb PCR pro ducts ob ta i ned from all se roty pes with cons tant se roty pe 1. This tech ni qu e was then mo di fi ed to re al-ti me flu o ro met ric nuc le ic acid de tec ti on system. R Re e s su ul lt ts s: : The PCR pro ducts ob ta i ned from seroty pes 1, 6A, 14 and 19F sho wed only ho mo dup lex bands whi le tho se from se roty pes 3, 5, 7F, 8, 9V, 11A, 15B and 23B sho wed spe ci fic he te ro dup lex bands. Alt ho ugh se roty pes 18C and 19A sho wed a spe ci fic he te ro dup lex ban ding pat tern, they co uld not be dif fe ren ti a ted from each ot her. When furt her he te ro dup lex analy sis was per for med to seroty pes 1, 6A, 14 and 19F with se roty pe 5 as a cons tant com po nent, se roty pe 1 and 6A co uld be dif fe ren ti a ted from the ot hers. He te ro dup lex and ho mo dup lex DNA strands co uld be dis tin gu is hed by mel ting cur ve analy sis in re al-time flu o ro met ric nuc le ic acid de tec ti on system. C Co on nc c l lu u s si i o on n: : As a ra pid and cost-ef fec ti ve met hod, re al-ti me he te rodup lex analy sis may be an al ter na ti ve to ot her mo le cu lar met hods for se roty pe pre dic ti on of pne u mo coc ci. K Ke ey y W Wo or rd ds s: : Streptococcus pneumoniae; serotyping; polymerase chain reaction; heteroduplex analysis Ö ÖZ ZE ET T A Am ma aç ç: : Li te ra tür de pnö mo kok se ro tip len dir me si ni da ha kul la nış lı ha le ge tir mek için bir kaç mo le kü ler sero tip tah min yön te mi ya yın lan mış tır. Bu ça lış ma nın ama cı bu mo le kü ler yak la şım la ra al ter na tif oluş tu ra cak farklı bir mo le kü ler se ro tip tah min yön te mi ge liş tir mek tir. G Ge e r re eç ç v ve e Y Yö ön n t te em m l le er r: : Tür ki ye' de en fek si yon lar dan en sık lık la izo le edi len S. pne u mo ni a e ser tip le ri olan 1, 3, 5, 6A, 7F, 8, 9V, 11A, 14, 15B, 18C, 19A, 19F ve 23B, ça -lış ma ya da hil edil miş tir. S. pne u mo ni a e kap sül po li sak ka ri ti nin iş len me si, dü zen len me si ve ta şın ma sı ile il gi li gen ler olan cpsA ve cpsB böl ge le ri PCR ile ço ğal tıl mış tır. El de edi len 1.8 kb bo yun da ki PCR ürün le ri he te ro dupleks ana li zi ne alın mış tır. Ana liz de tüm se ro tip le re a...

show abstract

Section: Discussionmentioning

confidence: 99%

Serotype Prediction for Frequently Isolated Serotypes of Streptococcus Pneumoniae by Heteroduplex Analysis and Modification of This Technique to Real-Time Fluorometric Nucleic Acid Detection System

Pinar¹,

Şener²,

Ustaçelebi³

2010

Turkiye Klinikleri J Med Sci

View full text Add to dashboard Cite

show abstract

“…It is an open chemistry platform that has 4–6 channel multiplexing capabilities and uses multiple excitation sources combined with several detection filters to detect virtually every known fluorophore. The change in the T m was considered an indicator of a mutation, and isolates for which the probe had a T m other than that for M. tuberculosis H37Rv was considered resistant to RFM [5]. The T m of wild rpoB is 73.8–74.8°C and that of mutant rpoB is 69.8–70.8°C.…”

Section: Methodsmentioning

confidence: 99%

Assessment of Status ofrpoBGene in FNAC Samples of Tuberculous Lymphadenitis by Real-Time PCR

Raoot

Dev

2012

Tuberculosis Research and Treatment

View full text Add to dashboard Cite

Introduction. Multidrug resistance tuberculosis (MDR TB), the combined resistance of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RFM) is a major public health problem in India as it ranks second among the MDR-TB high burden countries worldwide. WHO recommends RFM resistance as a “surrogate marker” for detecting MDR. FNAC is the most widely used noninvasive investigative technique for TB lymphadenitis. Real-time polymerase chain reaction, an extremely versatile technique can be used for the timely detection and treatment of MDR TB by assessing RFM resistance status in the FNAC samples of TB lymphadenitis. Aim. To assess the status of rpoB gene by real-time PCR in FNAC samples of TB lymphadenitis. Materials and Methods. Thirty FNAC samples from patients with persistent LAP or appearance of new LAP after 5 months or more of Anti Tubercular Treatment were assessed for status of rpoB gene by Real-Time PCR using probe covering the “hot spot resistance” region of the rpoB gene. Result. By using probe covering codons 531 and 526 of rpoB gene, we could detect 17 of 30 (56.7%) rifampin resistant isolate. The PCR could detect Mtb DNA in 100% of cases. Conclusion. Use of molecular methods like Real-Time PCR for detection of MDR-TB in FNAC samples is time saving, logical and economical approach over the culture based method.

show abstract

“…Moreover, since PCR detection does not require PCR product handling, nucleic acid contaminations attributable to PCR products are virtually eliminated. Regardless of these advantages, Real-Time-PCR amplification also presents an additional feature, much valuable for genotype analysis -the opportunity to perform melting curve analysis, and therefore to determine the melting temperature of a specific nucleic acid sequence (10)(11). The melting point determination is based on the fact that probe binding is a temperature and sequence dependent process.…”

Section: Introduction and Resultsmentioning

confidence: 99%

Advances in the genotyping of thrombosis genetic risk factors: clinical and laboratory implications

Cabeda¹,

Pereira²,

Oliveira³

et al. 2002

Pathophysiol Haemos Thromb

View full text Add to dashboard Cite

Mutation Detection by Fluorescent Hybridization Probe Melting Curves

Cited by 12 publications

References 26 publications

Serotype Prediction for Frequently Isolated Serotypes of Streptococcus Pneumoniae by Heteroduplex Analysis and Modification of This Technique to Real-Time Fluorometric Nucleic Acid Detection System

Serotype Prediction for Frequently Isolated Serotypes of Streptococcus Pneumoniae by Heteroduplex Analysis and Modification of This Technique to Real-Time Fluorometric Nucleic Acid Detection System

Assessment of Status ofrpoBGene in FNAC Samples of Tuberculous Lymphadenitis by Real-Time PCR

Advances in the genotyping of thrombosis genetic risk factors: clinical and laboratory implications

Contact Info

Product

Resources

About