Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital

Feb 22, 2016 - Digital PCR (dPCR) is a relatively new PCR technology that allows ..... from replicated reactions follows a log-normal distribution (wh...
2 downloads 0 Views 1MB Size
Subscriber access provided by La Trobe University Library

Article

Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR Dan Wang, Kevan M Yamahara, Yiping Cao, and Alexandria B. Boehm Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05747 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on February 28, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Environmental Science & Technology

Figure 1. The impact of gDNA fragmentation on the positive counts reported by dPCR. The error bars represent the Poisson 95% confidence interval resulted from 15,000-22,000 partitions per dPCR chip. (A1, gDNA extracted by Qiagen DNeasy blood and tissue kit for Gram-positive bacteria; A2, over-digested of A1 by HindIII B1 ~ B4, gDNA extracted by GeneRite DNA-EZ ST1 kit, for 1min (B1), 2.5 min (B2), 5 min (B3) and 10 min (B4) beadbeating, respectively). 114x79mm (300 x 300 DPI)

ACS Paragon Plus Environment

Environmental Science & Technology

Figure 2. dPCR quantification against nominal concentration derived from Qubit quantification. Error bars represent the Poisson 95% confidence interval for dPCR quantification. 85x79mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 2 of 40

Page 3 of 40

Environmental Science & Technology

Figure 3 qPCR and dPCR quantification for 24 environmental samples. Error bars represent the 95% confidence interval. Solid line is the linear regression line with zero intercept between qPCR and dPCR measurements. 105x100mm (300 x 300 DPI)

ACS Paragon Plus Environment

Environmental Science & Technology

Figure 4. The inhibitive effect of humic acid and calcium on qPCR (40 cycles reaction) and dPCR (45 cycles reaction). Error bars represent the 95% confidence interval. 155x141mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 4 of 40

Page 5 of 40

Environmental Science & Technology

Figure 5. Impact of thermal cycle numbers on dPCR quantification under influence of 5 ng/µl humic acid or 2 mM of calcium. Error bars represent the Poisson 95% confidence interval. 78x36mm (300 x 300 DPI)

ACS Paragon Plus Environment

Environmental Science & Technology

1

Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR

2 3

Dan Wang1,3*, Kevan M. Yamahara1, Yiping Cao2, Alexandria B. Boehm1,

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

1. Environmental and Water Studies, Department of Civil and Environmental Engineering, Stanford University, CA 94305, USA. 2. Southern California Coastal Water Research Project Authority, Costa Mesa, CA 92626, USA 3. Current address: California Department of Pesticide Regulation, Sacramento, CA 95812, USA

A manuscript for Environmental Science & Technology

*Corresponding author contact information: 1001 I Street, Sacramento, CA 95812. Phone: 916-324-4201. Email: [email protected].

ACS Paragon Plus Environment

Page 6 of 40

Page 7 of 40

Environmental Science & Technology

39

ABSTRACT

40

We evaluated the ability of chip-based digital PCR (dPCR) to quantify enterococci, the

41

fecal indicator recommended by USEPA for water quality monitoring. dPCR uses

42

Poisson statistics to estimate the number of DNA fragments in a sample with a specific

43

sequence. Underestimation may occur when a gene is redundantly encoded in the genome

44

and multiple copies of that gene are on one DNA fragment. When genomic DNA (gDNA)

45

was extracted using two commercial DNA extraction kits, we confirmed that dPCR could

46

discern individual copies of the redundant 23s rRNA gene in the enterococcal genome.

47

dPCR quantification was accurate when compared to the nominal concentration inferred

48

from fluorometer measurements (linear regression slope=0.98, intercept =0.03, R2 =0.99,

49

and p-value < 0.0001). dPCR quantification was also consistent with quantitative PCR

50

(qPCR) measurements as well as cell counts for BioBall® reference standard and 24

51

environmental water samples. qPCR and dPCR quantification of enterococci in the 24

52

environmental samples were significantly correlated (linear regression slope = 1.08, R2 of

53

0.96, and p-value 18 h later); therefore, rapid detection technologies with shorter sample-to-

68

result time are necessary to adequately protect public health.3-5 In 2012, the United States

69

Environmental Protection Agency (USEPA) published new recreational water quality

70

criteria that included numerical limits for enterococci enumerated using qPCR following

71

USEPA Method 16116 or other related qPCR methods.7 Thus, US laboratories

72

performing routine water quality monitoring have the option to enumerate enterococci

73

using qPCR.

74

Several limitations of qPCR have been reported that affect its ability to produce

75

accurate and consistent measurements across and within laboratories.8, 9 Quantification

76

via qPCR is relative; it is based on standard curves constructed from reference material

77

such as genomic DNA (gDNA), plasmid DNA, or synthesized DNA fragments.

78

Therefore, it requires careful calibration via the standard curve10, 11 and standardization of

79

the reference material across laboratories.12 Using a standard curve to accurately quantify

80

qPCR targets requires that the PCR amplification efficiency of target DNA in the

81

environmental samples be identical to the amplification efficiency of the reference

82

material. This requirement presents a challenge, as many environmental samples contain

83

PCR inhibitors that affect amplification efficiency.13, 14

ACS Paragon Plus Environment

Page 8 of 40

Page 9 of 40

Environmental Science & Technology

84

Digital PCR (dPCR) is a relatively new PCR technology that allows absolute

85

quantification of nucleic acids.15 In dPCR, the PCR mixture (typically 10-50 µl in volume)

86

is partitioned into many small individual reactions (typically 0.5-1.0 nl in volume). The

87

number of partitions needs to be large enough in order to separate individual copies of the

88

target sequences into different reactions; therefore, the number of partitions that are PCR-

89

positive reflects the copy numbers of the target sequence. Based on Poisson statistics, this

90

practice will ensure that the predominant of the individual partitions contain zero or one

91

copy of the target sequence (Poisson parameter λ should be