# Patent

## Title
Rapid quantification of butyric acid-producing bacteria using real-time PCR

## Abstract

A set of primer pairs for amplifying a nucleic acid of a butyric acid-producing bacteria, each primer containing an oligonucleotide selected from the buk gene region of the butyric acid-producing bacteria. The primers are particularly suitable for amplifying the nucleic acid of butyric acid-producing bacteria present in gas and oil production operations.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method for the rapid detection and quantification of butyric acid-producing bacteria using real-time quantitative polymerase chain reactions (qPCR).
2. Description of Related Art
Microbiologically influenced corrosion is a significant problem affecting the gas and oil industry as well as other industries, and the monitoring of microbial populations within such industries is a component of overall corrosion control operations. Traditionally, the monitoring of microbial populations in samples obtained from gas and oil production operations has employed microbial growth tests. In such tests, samples are diluted to various levels and used to inoculate microbial growth media that is designed to favor the growth of various types of bacteria. After days to several weeks of incubation, the growth tests are scored based on the presence or absence of growth in these various microbiological media. Unfortunately, as numerous researches show, only about 0.1% to about 10% bacteria from environmental samples can actually grow in an artificial medium, and a significant portion of bacteria growing in the media are not actually the target bacteria. Therefore, growth tests are unable to provide the accurate quantification of target bacteria in the samples.
To circumvent problems associated with such growth-based methods, many culture-independent genetic techniques have been developed in the past decade and applied for the detection of pathogens and quality control and assurance of products in the medicine and food industries. Because many ecosystems, including pipeline systems, have a relatively low abundance of microorganisms, the polymerase chain reaction (PCR) has been widely used to amplify the genetic signals of microbes in complex environmental samples. However, traditional PCR-based methods are significantly biased by amplification efficiency and the depletion of PCR reagents. Real-time quantitative PCR (qPCR) has been developed and used in the last few years in the medical and food research/industries to detect and quantify a number of pathogenic or infectious microorganisms. Quantitative PCR has also been used to determine the abundance of microorganisms in many different types of complex environmental samples such as sediments, water, wastewater, and marine samples. The advantage of quantitative PCR over traditional PCR is that it provides more accurate and reproducible quantification of microorganisms because quantitative PCR quantifies PCR products during the logarithmic phase of the reactions. Moreover, quantitative PCR offers a dynamic detection range of six orders of magnitude or more, does not need post-PCR manipulation, and has the capability of high throughput analysis.
Acid-producing bacteria are present in a variety of environments, including oil- and gas-bearing formations, soils, and domestic, industrial, and mining wastewaters. Acid-producing bacteria produce organic acids such as acetic, butyric, formatic, lactic, succinic, and propionic acids, and contribute to metal corrosion.
While the total bacterial number in an oil or gas production sample indicates the general conditions for bacteria to grow in this unique environment, and, thus, the potential risk of microbial corrosion, some specific groups of microorganisms commonly found in such samples probably play a more important role in the corrosion process. One such microorganism is a butyric acid-producing bacteria which has been found to be present in environmental samples and, in particular, samples from gas and oil production operations.
SUMMARY OF THE INVENTION
Accordingly, it is one object of this invention to provide a quantitative polymerase chain reaction assay that can be used to rapidly detect and quantify the butyric acid-producing bacteria in environmental samples, including, but not limited to, gas and oil production operations samples.
The most abundant acid-producing bacteria found in gas and oil production environments are acetic acid-producing bacteria and butyric acid-producing bacteria. The gene buk encodes the key enzyme butyrate kinase, which along with phosphotransbutyrylase, catalyzes the conversion of butyryl coenzyme A to butyrate. To address the above-stated object as well as other objects of this invention, a real-time qPCR assay has been developed by targeting the gene buk encoding butyrate kinase in butyric acid-producing bacteria. In one aspect, this invention features a set of PCR primer pairs that has been created to meet the specific requirements of real-time qPCR, which primer pairs amplify an approximately 103-bp DNA fragment from the buk gene of butyric acid-producing bacteria. The creation of the specific PCR primers comprising the primer pairs was accomplished by determining the DNA sequences of buk genes in butyric acid-producing bacteria most commonly found in gas and oil production operations. Each primer of the set of PCR primer pairs contains an oligonucleotide selected from the buk gene region of the butyric acid-producing bacteria. The set of PCR primer pairs comprises forward primers, buk-5F1 and buk-5F2, having oligonucleotides selected from the group of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and reverse primers, buk-6R1, buk-6R2, and buk-6R3, having oligonucleotides selected from the group of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.
The set of PCR primer pairs of this invention has been evaluated in SYBR Green I real-time qPCR on reference butyric acid-producing bacteria strains as well as gas pipeline samples. The primer combination gives the best detection and quantification of butyric acid-producing bacteria commonly found in the oil and gas production operations. In addition to being appropriate for the real-time PCR quantification of butyric acid-producing bacteria, the set of primers of this invention is specific for samples from gas and oil production operations and combines high throughput with high analytical sensitivity and precision, offering a dynamic detection range of 6 orders of magnitude or more.
In another aspect, this invention features a method for quantifying the amount of butyric acid-producing bacteria in a sample from a gas and/or oil production environment in which a nucleic acid in the genome of the butyric acid-producing bacteria is amplified by quantitative PCR to form a double-stranded nucleic acid product, the nucleic acid product is contacted with a label, such as a fluorophore, e.g. SYBR Green I, in a solution for binding therebetween, and the butyric acid-producing bacteria is quantified by monitoring the signal produced by the bound label, the intensity of which is a function of the quantity of the buk gene in butyric acid-producing bacteria in the sample.


DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
Definitions
As used herein, the term “gene” means a DNA sequence containing information required for expression of a polypeptide or protein.
As used herein, the term “primer” refers to a single-stranded oligonucleotide that is extended by covalent bonding of nucleotide monomers during amplification or polymerization of a nucleic acid molecule.
As used herein, the term “template” refers to a double-stranded or single-stranded nucleic acid molecule which is to be amplified, synthesized or sequenced. In the case of a double-stranded DNA molecule, denaturation of its strands to form a first and the second strand is performed before these molecules may be amplified, synthesized or sequenced. A primer, complementary to a portion of a template is hybridized under appropriate conditions and a polymerase then synthesizes a molecule complementary to the template or a portion thereof.
As used herein, the term “amplification” refers to any in vitro method for increasing the number of copies of a nucleotide sequence with the use of a DNA polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a DNA molecule or primer, thereby forming a new DNA molecule complementary to a DNA template. The formed DNA molecule and its template can be used as templates to synthesize additional DNA molecules. As used herein, one amplification reaction may consist of many rounds of DNA replication. DNA amplification reactions include polymerase chain reactions, PCR, which may consist of 10 to 50 cycles of denaturization and synthesis of a DNA molecule.
As used herein, the term “oligonucleotide” refers to a synthetic or natural molecule comprising a covalently linked sequence of nucleotides which are joined by a phosphodiester bond between the 3′ position of the pentose of one nucleotide and the 5′ position of the pentose of the adjacent nucleotide.
The invention disclosed herein is a PCR assay targeting the gene buk encoding butyrate kinase in butyric acid-producing bacteria using at least one of two novel forward primers, SEQ ID NO: 1 and SEQ ID NO: 2, and using at least one of three novel reverse primers, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The primers were designed based upon multiple alignments of partial sequences of the targeted buk gene retrieved from GenBank and gas pipeline samples. BioEdit Sequence Alignment Editor, Version 5.0.9, was used for multiple alignments of sequences, and manual searches for alignments were also conducted to determine candidate primer sequences. All primers were checked for hairpin structure and dimmers using a free Oligonucleotide Analyzer (available at www.rnature.com/oligonucleotide.html). MeltCal software, version 2.1 was then used to check the cross-hybridization among the sequences of primers used in some assay and calculate the melting temperature (Tm) using a nearest neighbor model. The sequences meet the general requirements of primer design for real-time quantitative PCR suggested by the RotoGene Real-Time PCR system manual provided by Corbett Research or in Inglis, G. D. et al., “Direct Quantification of Campylobacter jejuni and Campylobacter lanienae in Feces of Cattle by Real-Time Quantitative PCR”, Appl. Environ. Microbiol., 2004. 70(4): pp 2296-2306. In addition, all primer sequences were analyzed for specificity using a BLAST SEARCH FOR SHORT, NEARLY EXACT MATCHES program and were found not to cross-react with any other non-target organisms.
The PCR primer pairs of this invention are based upon the DNA sequences of buk genes from subsets of butyric acid-producing bacteria most commonly found in gas and oil production operations. Thus, the PCR primer pairs are ideally suited to accurately quantify butyric acid-producing bacteria in gas and oil production operations that have relevance to microbiologically influenced corrosion. If PCR primers were designed based on the DNA sequences of all known buk genes published in DNA sequence databases such as GenBank, the regions of conserved sequences that are suitable for use in designing PCR primers would be more limited. Primers designed on the entirety of all buk DNA sequences would by necessity fail to produce good yields from some buk genes.
Not all species of butyric acid-producing bacteria are commonly present in gas and oil production environments. Thus, PCR primers that target the sequences of those bacteria most commonly encountered in gas and oil production environments yield superior results in developing real-time PCR methods to accurately quantify butyric acid-producing bacteria in these samples and are capable of detecting the target butyric acid-producing bacteria with the highest possible sensitivity. The quantification using the real-time PCR method and primers of this invention is achieved by measuring the fluorescent signal of SYBR-Green I dye resulting from its specific binding to the double-stranded amplified fragments.
The target butyric acid-producing bacteria for which the set of PCR primers of this invention are a perfect match include Bacillus anthracis str. ‘Ames Ancestor’ (AE017334), Bacillus anthracis str. Ames (AE017037), Bacillus anthracis str. Sterne (AE017225), Bacillus cereus ATCC 10987 (AE017277), Bacillus cereus ATCC 14579 (AE017011), Bacillus cereus E33L (CP000001), Bacillus subtilis (Z99116), Bacillus thuringiensis serovar konkukian str. 97-27 (AE017355), Clostridium acetobutylicum ATCC 824 (AE007804), and Clostridium acetobutylicum butyrate kinase (L14744). All of these bacteria have the buk gene.
In general, the PCR amplification process employed in the method of this invention may be carried out using procedures known to those skilled in the art, namely, denaturing, annealing, and elongation, which can be repeated as many times as necessary to produce the desired amount of the target nucleic acid. While the number of cycles may be affected by any of a number of factors, such as the nature of the sample, the number of cycles required to achieve the desired amplification in accordance with this invention is generally in the range of about 10 to about 50. However, cycles numbers outside of this range may be necessary and, thus, the statement of the above stated range is in no way intended to limit the scope of this invention.
As previously indicated, the set of PCR primers of this invention comprises two forward primers, buk-5F1 (5′-CCA TGC ATT AAA TCA AAA AGC-3′, melting temperature (Tm): 56.5° C., SEQ ID NO: 1), buk-5F2 (5′-CCA TGC GTT AAA CCA AAA AGC-3′, Tm: 61.1° C., SEQ ID NO: 2), and three reverse primers buk-6R1 (5′-AGT ACC TCC ACC CAT GTG-3′, Tm: 59.6° C., SEQ ID NO: 3), buk-6R2 (5′-AAT ACC TCC GCC CAT ATG-3′, Tm: 57.5° C., SEQ ID NO: 4), buk-6R3 (5′-AAT ACC GCC RCC CAT ATG-3′, Tm: 58.1-61.4, SEQ ID NO: 5), all of which are perfect matches to the sequences of the above-mentioned butyric acid-producing bacteria. Thus, when this set of PCR primer pairs is used in the PCR amplification, only the above-mentioned butyric acid-producing bacteria are amplified. The primers, when used as pairs (at least one forward primer and at least one reverse primer), do not falsely amplify any bacteria which is not a butyric acid-producing bacteria. In addition, the primer pairs do not amplify sequences from Eukaryota, a high-level organism.
EXAMPLE
A q-PCR was performed using the primers of this invention, a Rotor-Gene 3000, 4 Channel Multiplexing, real-time PCR system (Corbett Research, Sydney, Australia), and a QuantiTect SYBR Green PCR kit (Qiagen, Inc., Valencia, Calif.). For quantification of the total bacteria, each 20 μL q-PCR reaction contained 1× QuantiTect SYBR Green PCR Master Mix, 800 nM forward primers (400 nM each of buk-5F1 and buk-5F2), 1400 nM reverse primers (400 nM buk-6R1; 200 nM buk-6R2; and 800 nM buk-6F3), one μL of template, and 4.6 μL water. The cycling conditions consisted of 15 minutes of incubation at 95° C. followed by 35 cycles of 95° C. for 30 sec, 52° C. for 30 sec, and 71° C. for denaturing, annealing and elongation, respectively, followed by 70° C. for 30 sec, and 70° C. to 99° C. to generate melting curves.
Using Clostridium acetobutylicum genomic DNA (ATCC 824) as a template, the linear amplification range for the quantification of the buk gene was 1×107 to 2×102 copies per reaction (correlation coefficient, R2>0.9999). The q-PCR product size was 103 bp from position 459 to 561 of Clostridium acetobutylicum ATCC 824 buk gene (complete genome GenBank Accession Number NC—003030, from 3224097 to 3226164).
While in the foregoing detailed description this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

## Claims

1. A set of primer pairs for amplifying a nucleic acid of a butyric acid-producing bacteria, each primer containing an oligonucleotide selected from the buk gene region of said butyric acid-producing bacteria, wherein the first primer is an oligonucleotide selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 and the second primer is an oligonucleotide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

2. The primer pair of claim 1, wherein said butyric acid-producing bacteria is selected from the group consisting of Bacillus anthracis str. ‘Ames Ancestor’ (Accession Number AE017334), Bacillus anthracis str. Ames (Accession Number AE017037), Bacillus anthracis str. Sterne (Accession Number AE017225), Bacillus cereus ATCC 10987 (Accession Number AE017277), Bacillus cereus ATCC 14579 (Accession Number AE017011), Bacillus cereus E33L (Accession Number CP000001), Bacillus subtilis (Accession Number Z99116), Bacillus thuringiensis serovar konkukian str. 97-27 (AE017355), Clostridium acetobutylicum ATCC 824 (Accession Number AE007804), Clostridium acetobutylicum butyrate kinase (Accession Number L14744), and mixtures thereof.

3. A PCR amplification method for quantification of butyric acid-producing bacteria comprising amplifying a nucleic acid of a butyric acid producing bacteria in the presence of forward primers selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. and mixtures thereof, reverse primers selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and mixtures thereof.

4. The PCR amplification method of claim 3, wherein said forward primers and said reverse primers comprise oligonucleotides selected from the buk gene region of said butyric acid-producing bacteria.

5. The PCR amplification method of claim 3, wherein nucleic acid produced by said method is quantified using a SYBR Green I dye.

6. The PCR amplification method of claim 3, wherein said butyric acid-producing bacteria is disposed in an environmental sample.

7. The PCR amplification method of claim 3, wherein said environmental sample is taken from at least one of a natural gas production operation and an oil production operation.