臺灣博碩士論文加值系統

敗血症(sepsis)是造成重症病患死亡的一個重要的疾病之一。在過去，血液培養是用來鑑定造成敗血症病原菌的黃金準則。而臨床上常無法有效培養分離出病原菌，分析其原因可歸因於以下幾點因素來討論：一、病原菌培養前病人已使用過抗生素；二、部份敗血症臨床症狀出現時，可能是由細胞激素活化所引起，而非病原菌本身；三、病原菌本身特性其生長速度緩慢不易培養。綜合上述因素，採集血液樣本的時機，是決定透過血液培養來鑑定出病原菌的重要因子之一。基於上述綜多可能影響血液培養的因素再加上血液培養時間經常需耗時48到72小時，對於病況危急病人臨床醫師只能使用經驗性抗生素( empiric antibiotics ) 治療模式治療病患，這也是造成抗生素濫用的因素之一。因此建立起快速可信賴的創新分子診斷工具來解決血液培養種種缺陷，對於臨床上將有重大貢獻，並且可以有效提供醫師選擇早期適合的抗生素使用，將有助於降低敗血症之死亡率。
而本研究可分成兩步驟來進行：
第一部分是建立創新分子診斷技術，我們發展出一套利用即時性聚合酶連鎖反應( real-time polymerase chain reaction, real-time PCR )結合高解析度融點分析( High-resolution melting analysis, HRMA )技術來進行臨床上常見25種病原菌的鑑定。利用ㄧ次高解析度融點分析即可鑑定九種臨床上常見病原菌，再經由異源雙鍵核酸分子行成反應( heteroduplex formation ) 可以有效鑑定出12種臨床上常見病原菌，最後再利用第二次即時性聚合酶連鎖反應可完成剩下4種病原菌的鑑定。
第二部分研究主要著眼於解決廣泛性即時性聚合酶連鎖反應(broad-range real-time PCR)目前無法有效解決的汙染問題。長久以來研究者藉由廣泛性即時性聚合酶連鎖反應的方法來鑑定病原菌一直受限於聚合酶 試劑製造時無法避免的細菌去氧核糖核酸(DNA)汙染而導致此技術在臨床上的應用受阻。因此本研究更進一步的透過引子擴增聚合酶連鎖反應 ( Primer extension PCR )的方式來有效降低試劑本身製造因素所造成的汙染問題，並且在不會產生偽陽性情況下，其偵測極限達10毫微微克。

Sepsis remains to be the leading cause of mortality in critical care patients. Early identification of causative pathogen in sepsis patients can improve clinical outcome, and the current gold standard is to use blood culture. However, blood culture is often not effective in identifying the pathogens in three common types of sepsis patients: (I) patients that have been recently treated with antibiotics before blood culture; (II) patients that have cytokine disorder instead of microbial infection; (III) patients that are infected with pathogens that are not easily cultured. Even if blood culture can identify the causative pathogen, it is rather time consuming, and often requires 48-72 hours to identify the microbial. Due to the above problems, clinicians often rely on empirical antibiotic treatment modalities for sepsis patients. This is because the risk of mortality increases substantially in hourly increment when the appropriate antimicrobial therapy is delayed. Although use of empirical antibiotic can be effective, it can instead cause an emergence of drug-resistant organisms.
Thus, my goal is to establish a rapid effective diagnostic tool for bloodstream infections, and thereby help clinicians select the most appropriate antibiotic treatment for sepsis patients. My research consists of two different parts. The first part is the establishment of a new innovative molecular diagnostic technique for microbial identification. To quantitatively identify microbial, I have combined real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) technology. Using slightly different approaches, I have successfully identified 25 clinical common pathogens using this platform: 9 bacterial species can be identified via a 1-step post-PCR high-resolution melting analysis; 12 bacterial species can be identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species; and 4 bacterial species can be identified by a 2nd real-time PCR targeting a different region of the 16S ribosomal ribonucleic acid (rRNA) gene.
The second part of my thesis is to solve bacterial deoxyribonucleic acid (DNA) contamination in PCR reagents. To solve contamination, we have employed broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. Broad-range PE-PCR amplification of the 16S rRNA gene can be validated and minute quantities of template DNA (10 femtogram) was detectable without false positives.

Table of Contents
指導教授推薦書
論文口試委員會審定書
誌謝 ..iii
中文摘要 ...iv
英文摘要 .vi
Table of Contents ..viii
List of Tables ..xi
List of Figures .xii
Chapter 1 Introduction……………………………………....……... 1
1.1 Importance of rapid infection diagnosis in surviving sepsis…………………………………………………….
1
1.2 Limitations of blood culture in sepsis diagnosis……….. 2
1.3 Need for improving current diagnostic tools for bloodstream infections……………………………….….
4
1.4 Bacterial load determination……………………………. 5
1.5 Molecular diagnostic techniques……………………….. 6
1.6 Amplification techniques for positive blood cultures…... 6
1.7 Amplification techniques for whole blood samples……. 7
1.8 Broad-range PCR assays………………………………... 8
1.9 16S rRNA Gene Sequencing for Bacterial Identification. 9
1.10 High resolution DNA melting analysis in clinical applications……………………………………….........
10
1.11 Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of 16S rRNA Amplicons…….
12
1.12 Challenges associated with 16S rRNA PCR………..…... 14
Chapter 2 Materials and Methods…………………………………. 17
2.1 Material………………………………………………… 17
2.2 Methods………………………………………………… 17
2.2.1 Isolation of bacterial genomic DNA……………………. 17
2.2.2 Pretreatment of samples prior to bacteria DNA isolation. 18
2.2.3 NucliSens Extractor…………………………………….. 19
2.2.4 Broad-range real-time PCR for amplification of 16s rRNA gene………………………..……………………..
19
2.2.5 Lightcycler Broad-range real-time PCR for amplification of 16s RNA gene…………………………
20
2.2.6 High-resolution melting-curve acquisition and analysis.. 20
2.2.7 Heteroduplex formation………………………………… 20
2.2.8 Broad-range primer extension-PCR…………………….. 21
2.2.9 Pretreatment of DNase I for decontamination and broad range PCR amplification…………….………………….
22
2.2.10 Agar Dilution Method for Quantitating Bacterial Colony-Forming Units Added to Whole-Blood Sample..
22
Chapter 3 Results………………………………………………....... 24
3.1 The melting profiles for detection of clinically Important Bacteria………………………………………
24
3.2 The high resolution melting analysis for the identification of the clinically Important Bacteria………
25
3.3 The contamination was a critical issue in broad-range amplification of bacterial DNA…………………………
30
3.4 Commercially available Taq DNA polymerase and PCR reagents are not sufficiently pure for broad-range bacterial DNA detection………………………………...

31
3.5 Decontamination bacteria DNA in PCR reagent by DNase I………………………………………………….
31
3.6 PE-PCR prevents co-amplification of contaminating DNA…………………………………………………….
32
3.7 Broad-range PE-PCR facilitates detection of minute quantities of bacterial DNA…..........................................
34
3.8 Broad-range real-time PE-PCR couples with HRMA for bacterial species identification………………………......
35
3.9 Comparison of broad-range real-time PE-PCR and broad-range real-time PCR with DNase I pretreatment of PCR reagents…………………………........................

36
3.10 Evaluation of DNA extraction protocol using blood culture samples………………………………………….
36
Chapter 4 Discussion and conclusion……………………………… 38
Reference……………………………………………….. 51
Tables……………………………………………70
Figures……………………………………………77


List of Tables
Table 1 The primers and fusion probes sequences……………… 70
Table 2 Melting peak profiles for clinically important bacterial strains disclosed by broad-range, real-time PCR of the 16S rRNA gene……………………………...…………..

72
Table 3 High-resolution melting profiles for clinically important bacterial species disclosed by broad-range 16S rRNA real-time PCR…………………………….......................

73
Table 4 The validation assay with 54 consecutive clinical bacteria isolates obtained from a clinical microbiology laboratory………………………..……………….……...

75
Table 5 High-resolution melting profiles for clinically important bacterial species disclosed by broad-range PE-PCR of 16S rRNA gene………………………………………….

76
List of Figures

Figure 1 Broad-range real-time PCR of bacterial genomic DNA and culture suspensions for high-resolution melting analysis by HR-1………………………………………...

77
Figure 2 High-resolution difference plots as the molecular fingerprints of 25 common and clinically important bacterial species…............................................................

78
Figure 3 Derivative plots for S. saprophyticus and S. bovis……... 80
Figure 4 Sequence alignments of the real-time PCR amplicons…. 81
Figure 5 High-resolution melting plots for the indicated bacterial species…………………………………………………..
82
Figure 6 Work flow for rapid detection and identification of 25 clinically important bacterial species……………………
83
Figure 7 Real-time PCR detection of bacterial 16S rRNA gene…. 85
Figure 8 HotStart and low-DNA Taq DNA polymerases are not sufficiently pure for sensitive and specific broad-range amplification of bacterial DNA…………………………

86
Figure 9 The principle of PE-PCR for bacterial DNA amplification and detection……………………………...
87
Figure 10 PE-PCR specifically amplifies template bacterial DNA without co-amplification of contaminating bacterial DNA……………………………………………………..

88
Figure 11 Comparison of broad-range real-time PE-PCR and broad-range real-time PCR with DNase I pretreatment of PCR reagents…………………………………………

90
Figure 12 Broad-range real-time PE-PCR and HRM analysis for 12 different bacterial species……………………………
92
Figure 13 High quality bacteria genomic DNA purified by DNA extraction protocol………………………………...…….
93
Figure 14 Sensitivity of the PCR assay in blood samples spiked with bacteria……………………………………………..
94

Anderson B. 1994. Broad-range polymerase chain reaction for detection and identification of bacteria. J Fla Med Assoc 81(12):835-837.
Anthony RM, Brown TJ, and French GL. 2000. Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array. J Clin Microbiol 38(2):781-788.
Antonelli M, and Mercurio G. 2009. The 2008 international guidelines for management of severe sepsis and septic shock: merits and weaknesses. Minerva Anestesiol 75(1-2):27-29.
Arpi M, Bentzon MW, Jensen J, and Frederiksen W. 1989. Importance of blood volume cultured in the detection of bacteremia. Eur J Clin Microbiol Infect Dis 8(9):838-842.
Avolio M, Diamante P, Zamparo S, Modolo ML, Grosso S, Zigante P, Tosoni N, De Rosa R, Stano P, and Camporese A. 2010. Molecular identification of bloodstream pathogens in patients presenting to the emergency department with suspected sepsis. Shock 34(1):27-30.
Bascunana CR, Mattsson JG, Bolske G, and Johansson KE. 1994. Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR. J Bacteriol 176(9):2577-2586.
Bloos F, Hinder F, Becker K, Sachse S, Mekontso Dessap A, Straube E, Cattoir V, Brun-Buisson C, Reinhart K, Peters G et al. . 2010. A multicenter trial to compare blood culture with polymerase chain reaction in severe human sepsis. Intensive Care Med 36(2):241-247.
Bosshard PP, Abels S, Zbinden R, Bottger EC, and Altwegg M. 2003. Ribosomal DNA sequencing for identification of aerobic gram-positive rods in the clinical laboratory (an 18-month evaluation). J Clin Microbiol 41(9):4134-4140.
Brakstad OG, Aasbakk K, and Maeland JA. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol 30(7):1654-1660.
Brosius J, Dull TJ, Sleeter DD, and Noller HF. 1981. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol 148(2):107-127.
Calandra T, and Cohen J. 2005. The international sepsis forum consensus conference on definitions of infection in the intensive care unit. Crit Care Med 33(7):1538-1548.
Carroll KC, Leonard RB, Newcomb-Gayman PL, and Hillyard DR. 1996. Rapid detection of the staphylococcal mecA gene from BACTEC blood culture bottles by the polymerase chain reaction. Am J Clin Pathol 106(5):600-605.
Carroll NM, Jaeger EE, Choudhury S, Dunlop AA, Matheson MM, Adamson P, Okhravi N, and Lightman S. 2000. Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR. J Clin Microbiol 38(5):1753-1757.
Champlot S, Berthelot C, Pruvost M, Bennett EA, Grange T, and Geigl EM. 2010. An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications. PLoS One 5(9).
Chapin K, and Musgnug M. 2003. Evaluation of three rapid methods for the direct identification of Staphylococcus aureus from positive blood cultures. J Clin Microbiol 41(9):4324-4327.
Cheng JC, Huang CL, Lin CC, Chen CC, Chang YC, Chang SS, and Tseng CP. 2006. Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clin Chem 52(11):1997-2004.
Cherkaoui A, Emonet S, Ceroni D, Candolfi B, Hibbs J, Francois P, and Schrenzel J. 2009. Development and validation of a modified broad-range 16S rDNA PCR for diagnostic purposes in clinical microbiology. J Microbiol Methods 79(2):227-231.
Chou LS, Lyon E, and Wittwer CT. 2005. A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning: cystic fibrosis transmembrane conductance regulator gene as a model. Am J Clin Pathol 124(3):330-338.
Cilia V, Lafay B, and Christen R. 1996. Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level. Mol Biol Evol 13(3):451-461.
Clarridge JE, 3rd. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17(4):840-862, table of contents.
Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, and Fox AJ. 2000. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 38(5):1747-1752.
Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R, Reinhart K, Angus DC, Brun-Buisson C, Beale R et al. . 2008. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 36(1):296-327.
Dierkes C, Ehrenstein B, Siebig S, Linde HJ, Reischl U, and Salzberger B. 2009. Clinical impact of a commercially available multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis. BMC Infect Dis 9:126.
Erali M, Voelkerding KV, and Wittwer CT. 2008. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85(1):50-58.
Fenollar F, Levy PY, and Raoult D. 2008. Usefulness of broad-range PCR for the diagnosis of osteoarticular infections. Curr Opin Rheumatol 20(4):463-470.
Fenollar F, and Raoult D. 2007. Molecular diagnosis of bloodstream infections caused by non-cultivable bacteria. Int J Antimicrob Agents 30 Suppl 1:S7-15.
Fenollar F, Roux V, Stein A, Drancourt M, and Raoult D. 2006. Analysis of 525 samples to determine the usefulness of PCR amplification and sequencing of the 16S rRNA gene for diagnosis of bone and joint infections. J Clin Microbiol 44(3):1018-1028.
Fraser A, Paul M, Almanasreh N, Tacconelli E, Frank U, Cauda R, Borok S, Cohen M, Andreassen S, Nielsen AD et al. . 2006. Benefit of appropriate empirical antibiotic treatment: thirty-day mortality and duration of hospital stay. Am J Med 119(11):970-976.
Fredricks DN, and Relman DA. 1999. Application of polymerase chain reaction to the diagnosis of infectious diseases. Clin Infect Dis 29(3):475-486; quiz 487-478.
Fujita S, Senda Y, Iwagami T, and Hashimoto T. 2005. Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis. J Clin Microbiol 43(3):1149-1157.
Furet JP, Quenee P, and Tailliez P. 2004. Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR. Int J Food Microbiol 97(2):197-207.
Garnacho-Montero J, Garcia-Garmendia JL, Barrero-Almodovar A, Jimenez-Jimenez FJ, Perez-Paredes C, and Ortiz-Leyba C. 2003. Impact of adequate empirical antibiotic therapy on the outcome of patients admitted to the intensive care unit with sepsis. Crit Care Med 31(12):2742-2751.
Gebert S, Siegel D, and Wellinghausen N. 2008. Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis. J Infect 57(4):307-316.
Glerant JC, Hellmuth D, Schmit JL, Ducroix JP, and Jounieaux V. 1999. Utility of blood cultures in community-acquired pneumonia requiring hospitalization: influence of antibiotic treatment before admission. Respir Med 93(3):208-212.
Glushkov SA, Bragin AG, and Dymshits GM. 2009. Decontamination of polymerase chain reaction reagents using DEAE-cellulose. Anal Biochem 393(1):135-137.
Graham R, Liew M, Meadows C, Lyon E, and Wittwer CT. 2005. Distinguishing different DNA heterozygotes by high-resolution melting. Clin Chem 51(7):1295-1298.
Greisen K, Loeffelholz M, Purohit A, and Leong D. 1994. PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 32(2):335-351.
Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, and Wittwer CT. 2003. Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem 49(3):396-406.
Gurtler V, and Stanisich VA. 1996. New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiology 142 ( Pt 1):3-16.
Hall KK, and Lyman JA. 2006. Updated review of blood culture contamination. Clin Microbiol Rev 19(4):788-802.
Hansen WL, Bruggeman CA, and Wolffs PF. 2009. Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood. J Clin Microbiol 47(8):2629-2631.
Hendolin PH, Markkanen A, Ylikoski J, and Wahlfors JJ. 1997. Use of multiplex PCR for simultaneous detection of four bacterial species in middle ear effusions. J Clin Microbiol 35(11):2854-2858.
Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, and Voelkerding KV. 2006. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem 52(3):494-503.
Herrmann MG, Durtschi JD, Bromley LK, Wittwer CT, and Voelkerding KV. 2007a. Instrument comparison for heterozygote scanning of single and double heterozygotes: a correction and extension of Herrmann et al., Clin Chem 2006;52:494-503. Clin Chem 53(1):150-152.
Herrmann MG, Durtschi JD, Wittwer CT, and Voelkerding KV. 2007b. Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem 53(8):1544-1548.
Hicks P, Cooper DJ, Webb S, Myburgh J, Seppelt I, Peake S, Joyce C, Stephens D, Turner A, French C et al. . 2008. The Surviving Sepsis Campaign: International guidelines for management of severe sepsis and septic shock: 2008. An assessment by the Australian and New Zealand intensive care society. Anaesth Intensive Care 36(2):149-151.
Hiratsuka M, Narahara K, Kishikawa Y, Ismail Hamdy S, Endo N, Agatsuma Y, Matsuura M, Inoue T, Tomioka Y, and Mizugaki M. 2002. A simultaneous LightCycler detection assay for five genetic polymorphisms influencing drug sensitivity. Clin Biochem 35(1):35-40.
Hughes MS, Beck LA, and Skuce RA. 1994. Identification and elimination of DNA sequences in Taq DNA polymerase. J Clin Microbiol 32(8):2007-2008.
Hung CC, Lee CN, Chang CH, Jong YJ, Chen CP, Hsieh WS, Su YN, and Lin WL. 2008. Genotyping of the G1138A mutation of the FGFR3 gene in patients with achondroplasia using high-resolution melting analysis. Clin Biochem 41(3):162-166.
Hurtle W, Shoemaker D, Henchal E, and Norwood D. 2002. Denaturing HPLC for identifying bacteria. Biotechniques 33(2):386-388, 390-381.
Jensen MA, Webster JA, and Straus N. 1993. Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl Environ Microbiol 59(4):945-952.
Johnson JR. 2000. Development of polymerase chain reaction-based assays for bacterial gene detection. J Microbiol Methods 41(3):201-209.
Jordan JA, Jones-Laughner J, and Durso MB. 2009. Utility of pyrosequencing in identifying bacteria directly from positive blood culture bottles. J Clin Microbiol 47(2):368-372.
Kattar MM, Chavez JF, Limaye AP, Rassoulian-Barrett SL, Yarfitz SL, Carlson LC, Houze Y, Swanzy S, Wood BL, and Cookson BT. 2000. Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia. J Clin Microbiol 38(2):789-794.
Klappenbach JA, Saxman PR, Cole JR, and Schmidt TM. 2001. rrndb: the Ribosomal RNA Operon Copy Number Database. Nucleic Acids Res 29(1):181-184.
Klausegger A, Hell M, Berger A, Zinober K, Baier S, Jones N, Sperl W, and Kofler B. 1999. Gram type-specific broad-range PCR amplification for rapid detection of 62 pathogenic bacteria. J Clin Microbiol 37(2):464-466.
Klouche M, and Schroder U. 2008. Rapid methods for diagnosis of bloodstream infections. Clin Chem Lab Med 46(7):888-908.
Kollef MH, Sherman G, Ward S, and Fraser VJ. 1999. Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients. Chest 115(2):462-474.
Kotilainen P, Jalava J, Meurman O, Lehtonen OP, Rintala E, Seppala OP, Eerola E, and Nikkari S. 1998. Diagnosis of meningococcal meningitis by broad-range bacterial PCR with cerebrospinal fluid. J Clin Microbiol 36(8):2205-2209.
Krypuy M, Ahmed AA, Etemadmoghadam D, Hyland SJ, DeFazio A, Fox SB, Brenton JD, Bowtell DD, and Dobrovic A. 2007. High resolution melting for mutation scanning of TP53 exons 5-8. BMC Cancer 7:168.
Lamoth F, Jaton K, Prod'hom G, Senn L, Bille J, Calandra T, and Marchetti O. 2010. Multiplex blood PCR in combination with blood cultures for improvement of microbiological documentation of infection in febrile neutropenia. J Clin Microbiol 48(10):3510-3516.
Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, and Wittwer C. 2004. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50(7):1156-1164.
Lin JH, Tseng CP, Chen YJ, Lin CY, Chang SS, Wu HS, and Cheng JC. 2008. Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. J Clin Microbiol 46(3):1090-1097.
Mackay JF, Wright CD, and Bonfiglioli RG. 2008. A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods 4:8.
Maubon D, Hamidfar-Roy R, Courby S, Vesin A, Maurin M, Pavese P, Ravanel N, Bulabois CE, Brion JP, Pelloux H et al. . 2010. Therapeutic impact and diagnostic performance of multiplex PCR in patients with malignancies and suspected sepsis. J Infect 61(4):335-342.
McCabe KM, Zhang YH, Huang BL, Wagar EA, and McCabe ER. 1999. Bacterial species identification after DNA amplification with a universal primer pair. Mol Genet Metab 66(3):205-211.
McKenzie R, and Reimer LG. 1987. Effect of antimicrobials on blood cultures in endocarditis. Diagn Microbiol Infect Dis 8(3):165-172.
Meier A, Persing DH, Finken M, and Bottger EC. 1993. Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens. J Clin Microbiol 31(3):646-652.
Mermel LA, and Maki DG. 1993. Detection of bacteremia in adults: consequences of culturing an inadequate volume of blood. Ann Intern Med 119(4):270-272.
Michel F, Lazowska J, Faye G, Fukuhara H, and Slonimski PP. 1974. Physical and genetic organization of Petite and Grande yeast mitochondrial DNA. III. High resolution melting and reassociation studies. J Mol Biol 85(3):411-431.
Millar BC, Xu J, and Moore JE. 2002. Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J Clin Microbiol 40(5):1575-1580.
Millar BC, Xu J, and Moore JE. 2007. Molecular diagnostics of medically important bacterial infections. Curr Issues Mol Biol 9(1):21-39.
Montgomery JL, Sanford LN, and Wittwer CT. 2010. High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev Mol Diagn 10(2):219-240.
Munson EL, Diekema DJ, Beekmann SE, Chapin KC, and Doern GV. 2003. Detection and treatment of bloodstream infection: laboratory reporting and antimicrobial management. J Clin Microbiol 41(1):495-497.
Neefs JM, Van de Peer Y, De Rijk P, Chapelle S, and De Wachter R. 1993. Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res 21(13):3025-3049.
Odell ID, Cloud JL, Seipp M, and Wittwer CT. 2005. Rapid species identification within the Mycobacterium chelonae-abscessus group by high-resolution melting analysis of hsp65 PCR products. Am J Clin Pathol 123(1):96-101.
Bizzini A, Greub G. 2010. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification. Clin Microbiol Infect 16: 1614–1619.
Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, et al. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318: 283–287.
Buckwalter MR, Nga PT, Gouilh MA, Fiette L, Bureau JF, et al. (2011) Identification of a Novel Neuropathogenic Theiler’s Murine Encephalomyelitis Virus. J Virol 85: 6893–6905.
Nakamura S, Maeda N, Miron IM, Yoh M, Izutsu K, et al. (2008) Metagenomic diagnosis of bacterial infections. Emerg Infect Dis 14: 1784–1786.
Nakamura S, Nakaya T, Iida T (2011) Metagenomic analysis of bacterial infections by means of high-throughput DNA sequencing. Exp Biol Med (Maywood) 236: 968–971.
Oliveira K, Procop GW, Wilson D, Coull J, and Stender H. 2002. Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes. J Clin Microbiol 40(1):247-251.
Ou CY, Moore JL, and Schochetman G. 1991. Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques 10(4):442, 444, 446.
Palais R, and Wittwer CT. 2009. Mathematical algorithms for high-resolution DNA melting analysis. Methods Enzymol 454:323-343.
Pandit L, Kumar S, and Karunasagar I. 2005. Diagnosis of partially treated culture-negative bacterial meningitis using 16S rRNA universal primers and restriction endonuclease digestion. J Med Microbiol 54(Pt 6):539-542.
Patel JB. 2001. 16S rRNA gene sequencing for bacterial pathogen identification in the clinical laboratory. Mol Diagn 6(4):313-321.
Peters RP, van Agtmael MA, Danner SA, Savelkoul PH, and Vandenbroucke-Grauls CM. 2004. New developments in the diagnosis of bloodstream infections. Lancet Infect Dis 4(12):751-760.
Petershofen EK, Fislage R, Faber R, Schmidt H, Roth WK, and Seifried E. 2000. Detection of nucleic acid sequences from bacterial species with molecular genetic methods. Transfus Sci 23(1):21-27.
Pettersson B, Johansson KE, and Uhlen M. 1994. Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing. Appl Environ Microbiol 60(7):2456-2461.
Pletz MW, Wellinghausen N, and Welte T. 2011. Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view. Intensive Care Med 37(7):1069-1076.
Qian Q, Tang YW, Kolbert CP, Torgerson CA, Hughes JG, Vetter EA, Harmsen WS, Montgomery SO, Cockerill FR, 3rd, and Persing DH. 2001. Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results. J Clin Microbiol 39(10):3578-3582.
Radstrom P, Backman A, Qian N, Kragsbjerg P, Pahlson C, and Olcen P. 1994. Detection of bacterial DNA in cerebrospinal fluid by an assay for simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and streptococci using a seminested PCR strategy. J Clin Microbiol 32(11):2738-2744.
Reed GH, Kent JO, and Wittwer CT. 2007. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8(6):597-608.
Reed GH, and Wittwer CT. 2004. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem 50(10):1748-1754.
Rello J, Quintana E, Mirelis B, Gurgui M, Net A, and Prats G. 1993. Polymicrobial bacteremia in critically ill patients. Intensive Care Med 19(1):22-25.
Reno WL, 3rd, McDaniel DO, Turner WW, Jr., and Williams MD. 2001. Polymerase chain reaction for the detection of bacteremia. Am Surg 67(6):508-512.
Ririe KM, Rasmussen RP, and Wittwer CT. 1997. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245(2):154-160.
Robertson T, Bibby S, O'Rourke D, Belfiore T, Lambie H, and Noormohammadi AH. 2009. Characterization of Chlamydiaceae species using PCR and high resolution melt curve analysis of the 16S rRNA gene. J Appl Microbiol 107(6):2017-2028.
Sacchi CT, Whitney AM, Reeves MW, Mayer LW, and Popovic T. 2002. Sequence diversity of Neisseria meningitidis 16S rRNA genes and use of 16S rRNA gene sequencing as a molecular subtyping tool. J Clin Microbiol 40(12):4520-4527.
Sachse S, Straube E, Lehmann M, Bauer M, Russwurm S, and Schmidt KH. 2009. Truncated human cytidylate-phosphate-deoxyguanylate-binding protein for improved nucleic acid amplification technique-based detection of bacterial species in human samples. J Clin Microbiol 47(4):1050-1057.
Schaefer S, Glebe D, Wend UC, Oyunbileg J, and Gerlich WH. 2003. Universal primers for real-time amplification of DNA from all known Orthohepadnavirus species. J Clin Virol 27(1):30-37.
Schmid KJ, Sorensen TR, Stracke R, Torjek O, Altmann T, Mitchell-Olds T, and Weisshaar B. 2003. Large-scale identification and analysis of genome-wide single-nucleotide polymorphisms for mapping in Arabidopsis thaliana. Genome Res 13(6A):1250-1257.
Seipp MT, Durtschi JD, Voelkerding KV, and Wittwer CT. 2009. Multiplex amplicon genotyping by high-resolution melting. J Biomol Tech 20(3):160-164.
Serody JS, Berrey MM, Albritton K, O'Brien SM, Capel EP, Bigelow SH, Weber DJ, Gabriel, Wiley JM, Schell MJ et al. . 2000. Utility of obtaining blood cultures in febrile neutropenic patients undergoing bone marrow transplantation. Bone Marrow Transplant 26(5):533-538.
Silkie SS, Tolcher MP, and Nelson KL. 2008. Reagent decontamination to eliminate false-positives in Escherichia coli qPCR. J Microbiol Methods 72(3):275-282.
Sleigh J, Cursons R, and La Pine M. 2001. Detection of bacteraemia in critically ill patients using 16S rDNA polymerase chain reaction and DNA sequencing. Intensive Care Med 27(8):1269-1273.
Spangler R, Goddard NL, and Thaler DS. 2009. Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria. PLoS One 4(9):e7010.
Steinert M, and Van Assel S. 1974. Base compisition heterogeneity in kinetoplast DNA FROM FOUR SPECIES OF HEMOFLAGELLATES. Biochem Biophys Res Commun 61(4):1249-1255.
Steinman CR, Muralidhar B, Nuovo GJ, Rumore PM, Yu D, and Mukai M. 1997. Domain-directed polymerase chain reaction capable of distinguishing bacterial from host DNA at the single-cell level: characterization of a systematic method to investigate putative bacterial infection in idiopathic disease. Anal Biochem 244(2):328-339.
Stephens AJ, Inman-Bamber J, Giffard PM, and Huygens F. 2008. High-resolution melting analysis of the spa repeat region of Staphylococcus aureus. Clin Chem 54(2):432-436.
Tajiri-Utagawa E, Hara M, Takahashi K, Watanabe M, and Wakita T. 2009. Development of a rapid high-throughput method for high-resolution melting analysis for routine detection and genotyping of noroviruses. J Clin Microbiol 47(2):435-440.
Tan AY, Westerman DA, Carney DA, Seymour JF, Juneja S, and Dobrovic A. 2008. Detection of NPM1 exon 12 mutations and FLT3 - internal tandem duplications by high resolution melting analysis in normal karyotype acute myeloid leukemia. J Hematol Oncol 1:10.
Tan TY, Corden S, Barnes R, and Cookson B. 2001. Rapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR. J Clin Microbiol 39(12):4529-4531.
Toyota T, Watanabe A, Shibuya H, Nankai M, Hattori E, Yamada K, Kurumaji A, Karkera JD, Detera-Wadleigh SD, and Yoshikawa T. 2000. Association study on the DUSP6 gene, an affective disorder candidate gene on 12q23, performed by using fluorescence resonance energy transfer-based melting curve analysis on the LightCycler. Mol Psychiatry 5(5):489-494.
Tseng CP, Cheng JC, Tseng CC, Wang C, Chen YL, Chiu DT, Liao HC, and Chang SS. 2003. Broad-range ribosomal RNA real-time PCR after removal of DNA from reagents: melting profiles for clinically important bacteria. Clin Chem 49(2):306-309.
Turenne CY, Witwicki E, Hoban DJ, Karlowsky JA, and Kabani AM. 2000. Rapid identification of bacteria from positive blood cultures by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J Clin Microbiol 38(2):513-520.
Valles J, Rello J, Ochagavia A, Garnacho J, and Alcala MA. 2003. Community-acquired bloodstream infection in critically ill adult patients: impact of shock and inappropriate antibiotic therapy on survival. Chest 123(5):1615-1624.
Vaughn CP, and Elenitoba-Johnson KS. 2004. High-resolution melting analysis for detection of internal tandem duplications. J Mol Diagn 6(3):211-216.
von Lilienfeld-Toal M, Lehmann LE, Raadts AD, Hahn-Ast C, Orlopp KS, Marklein G, Purr I, Cook G, Hoeft A, Glasmacher A et al. . 2009. Utility of a commercially available multiplex real-time PCR assay to detect bacterial and fungal pathogens in febrile neutropenia. J Clin Microbiol 47(8):2405-2410.
Wallet F, Nseir S, Baumann L, Herwegh S, Sendid B, Boulo M, Roussel-Delvallez M, Durocher AV, and Courcol RJ. 2010. Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood. Clin Microbiol Infect 16(6):774-779.
Weinstein MP, Murphy JR, Reller LB, and Lichtenstein KA. 1983a. The clinical significance of positive blood cultures: a comprehensive analysis of 500 episodes of bacteremia and fungemia in adults. II. Clinical observations, with special reference to factors influencing prognosis. Rev Infect Dis 5(1):54-70.
Weinstein MP, Reller LB, Murphy JR, and Lichtenstein KA. 1983b. The clinical significance of positive blood cultures: a comprehensive analysis of 500 episodes of bacteremia and fungemia in adults. I. Laboratory and epidemiologic observations. Rev Infect Dis 5(1):35-53.
Wellinghausen N, Wirths B, Essig A, and Wassill L. 2004a. Evaluation of the Hyplex BloodScreen Multiplex PCR-Enzyme-linked immunosorbent assay system for direct identification of gram-positive cocci and gram-negative bacilli from positive blood cultures. J Clin Microbiol 42(7):3147-3152.
Wellinghausen N, Wirths B, Franz AR, Karolyi L, Marre R, and Reischl U. 2004b. Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes. Diagn Microbiol Infect Dis 48(4):229-241.
White HE, Hall VJ, and Cross NC. 2007. Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes. Clin Chem 53(11):1960-1962.
Wilbrink B, van der Heijden IM, Schouls LM, van Embden JD, Hazes JM, Breedveld FC, and Tak PP. 1998. Detection of bacterial DNA in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers. Arthritis Rheum 41(3):535-543.
Wilck MB, Wu Y, Howe JG, Crouch JY, and Edberg SC. 2001. Endocarditis caused by culture-negative organisms visible by Brown and Brenn staining: utility of PCR and DNA sequencing for diagnosis. J Clin Microbiol 39(5):2025-2027.
Wilson KH, Blitchington RB, and Greene RC. 1990. Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J Clin Microbiol 28(9):1942-1946.
Wittwer CT. 2009. High-resolution DNA melting analysis: advancements and limitations. Hum Mutat 30(6):857-859.
Wittwer CT, Herrmann MG, Moss AA, and Rasmussen RP. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22(1):130-131, 134-138.
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, and Pryor RJ. 2003. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49(6 Pt 1):853-860.
Woese CR, Stackebrandt E, Macke TJ, and Fox GE. 1985. A phylogenetic definition of the major eubacterial taxa. Syst Appl Microbiol 6:143-151.
Wojdacz TK, and Dobrovic A. 2007. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 35(6):e41.
Won H, Rothman R, Ramachandran P, Hsieh YH, Kecojevic A, Carroll KC, Aird D, Gaydos C, and Yang S. 2010. Rapid identification of bacterial pathogens in positive blood culture bottles by use of a broad-based PCR assay coupled with high-resolution melt analysis. J Clin Microbiol 48(9):3410-3413.
Woo PC, Fung AM, Lau SK, and Yuen KY. 2002. Identification by 16S rRNA gene sequencing of Lactobacillus salivarius bacteremic cholecystitis. J Clin Microbiol 40(1):265-267.
Woo PC, Lau SK, Teng JL, Tse H, and Yuen KY. 2008. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 14(10):908-934.
Yagupsky P, and Nolte FS. 1990. Quantitative aspects of septicemia. Clin Microbiol Rev 3(3):269-279.
Yang S, Ramachandran P, Rothman R, Hsieh YH, Hardick A, Won H, Kecojevic A, Jackman J, and Gaydos C. 2009. Rapid identification of biothreat and other clinically relevant bacterial species by use of universal PCR coupled with high-resolution melting analysis. J Clin Microbiol 47(7):2252-2255.
Zhou L, Vandersteen J, Wang L, Fuller T, Taylor M, Palais B, and Wittwer CT. 2004. High-resolution DNA melting curve analysis to establish HLA genotypic identity. Tissue Antigens 64(2):156-164.
Zhou L, Wang L, Palais R, Pryor R, and Wittwer CT. 2005. High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem 51(10):1770-1777.