Sensitive detection of bacteria by improved nested polymerase chain reaction targeting the 16S ribosomal RNA gene and identification of bacterial species by amplicon sequencing

Description

RELATED APPLICATIONS

The present application is a continuation of U.S. patent application Ser. No. 11/204,854, filed Aug. 15, 2005, which claims benefit of priority from U.S. Provisional Patent Application 60/603,120, filed Aug. 20, 2004, each of which is expressly incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of PCR methods, and specific primers therefore, as well as their use in the identification of any type of bacteria, and in particular RNA forms of bacteria.

BACKGROUND OF THE INVENTION

The use of biological fluids for therapeutic human application such as plasmas, albumin, live vaccines, stem cells requires that they are absolutely devoid of bacterial contamination. It has been found that filtering, and possibly other traditional methods, may fail to eliminate all forms of organisms, leading to possible contamination or experimental artifacts.

It is believed that certain pathologies are associated with bacteria or bacterial forms which are difficult to detect, and which may pass through nano-porous barriers. This leads to possible errors in diagnosis or causation, and which may lead to erroneous treatment and impede prevention.

See, Hopert, Anne, Uphoff, Cord C., Wirth, Manfred, Hauser, Hansjorg, and Drexler, Hans G., “Specificity and sensitivity of Polymerase Chain Reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines”, J. Immunological Methods, 164 (1993):91-100, expressly incorporated herein by reference.

Kajander, E. O., et al., “Comparison of Staphylococci and novel Bacteria-Like Particles from blood”, Zbl. Bakt. Suppl. 26, 1994, expressly incorporated herein by reference.

Akerman, Kari K., “Scanning Electron Microscopy of Nanobacteria—Novel Biofilm Producing Organisms in Blood”, Scanning Vol. 15, Suppl. III (1993), expressly incorporated herein by reference. www.newcastle.edu.au/discipline/biology/projects/hons_cpru.html, expressly incorporated herein by reference.

Cifticioglu, Neva, et al., “Apoptotic effect of nanobacteria on cultured mammalian cells”, Mol. Biol. Cell. Suppl., Vol. 7 (1996):517a

Cifticioglu, Neva, et al., “A new potential threat in antigen and antibody products: Nanobacteria”, Vaccines 97, Brown et al. Ed., Cold Spring Harbor Laboratory Press, New York, 1997, expressly incorporated herein by reference.

Baseman, Joel B., et al., “Mycoplasmas: Sophisticated, Reemerging, and Burdened by their Notoriety”, EID Vol. 3, N^o 1 www.cdc.gov/ncidod/EID/vol3nol/baseman.htm, expressly incorporated herein by reference.

Relman, David A., “Detection and Identification of Previously Unrecognized Microbial Pathogens”, EID Vol. 4, N^o 3 www.cdc.gov/ncidod/EID/vol4no3/relman.htm, expressly incorporated herein by reference.

Mattman, Lida H., Cell Wall Deficient Forms-Stealth Pathogens, 2nd Ed., CRC Press (1993), expressly incorporated herein by reference.

U.S. Pat. No. 5,688,646, expressly incorporated herein by reference, describes novel mycoplasmas which are prominent in patients who are thought to be suffering from AIDS. Devices are also provided for the in vitro detection of mycoplasmas in biological fluid by means of a reagent which is specific for the mycoplasma group without being specific for particular species within said group. Devices for testing mycoplasma sensitivity to antibiotics are also described.

See the following, each of which and cited references is expressly incorporated herein by reference in their entirety:

Pat. No	Title

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6,228,584	DexB
6,210,880	Polymorphism analysis by nucleic acid structure probing with structure-bridging
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6,204,014	DnaB
6,197,549	Ama
6,197,300	ftsZ
6,194,170	MurF of Streptococcus pneumoniae
6,190,881	Ribonucleotide diphosphate reductase, nrdF, of streptococcus pneumoniae
6,168,797	FabF
6,165,992	Histidine kinase
6,165,991	Sensor histidine kinase of Streptococcus pneumoniae
6,165,764	Polynucleotides encoding tRNA methyl transferases from Streptococcus pneumoniae
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6,156,537	Phospho-N-acetylmuramoyl-pentapeptide transferase of Streptococcus pneumoniae
6,146,863	Staphylococcus aureus 3-hydroxyacyl-CoA dehydrogenase
6,146,846	Primosome protein a of streptococcus pneumoniae
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5,763,246	DNA encoding arginyl tRNA synthetase from staphylococcus aureus
5,750,387	DNA encoing leucyl TRNA synthetase from staphylococcus aureus
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5,919,625	Ribonuclease resistant viral RNA standards
5,912,117	Method for diagnosis of lyme disease
5,907,085	Grapevine leafroll virus proteins and their uses
5,876,924	Nucleic acid amplification method hybridization signal amplification method
	(HSAM)
5,843,669	Cleavage of nucleic acid acid using thermostable methoanococcus jannaschii FEN-1
	endonucleases
5,830,670	Neural thread protein gene expression and detection of Alzheimer's disease
5,795,976	Detection of nucleic acid heteroduplex molecules by denaturing high-performance
	liquid chromatography and methods for comparative sequencing
5,763,169	Nucleic acid probes for the detection and identification of fungi
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5,744,306	Methods for nucleic acid detection, sequencing, and cloning using exonuclease
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20030124545	Quantitative assay for the simultaneous detection and speciation of bacterial
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20030017532	ndp
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20020160447	Ups (ugc)
20020150963	Map
20020146790	MurF
20020146751	fabF
20020128437	GidA1
20020127596	murF2
20020123047	dexB
20020120116	Enterococcus faecalis polynucleotides and polypeptides
20020119520	FabZ
20020119510	gcp
20020119454	Polymorphism analysis by nucleic acid structure probing with structure-bridging
	oligonucleotides
20020115075	nadE
20020106776	pth
20020102700	metK
20020098544	nrdG
20020091237	nusB
20020082234	Novel prokaryotic polynucleotides, polypeptides and their uses
20020058799	Novel pth
20020052472	ama
20020048789	birA
20020025516	NRDE
20020004581	gcp
20020004580	ftsZ
20010027183	tdk
20010023064	yfjO
20010020010	Histidine kinase
20010016334	MurF
20010014670	Treatment and diagnosis of Alzheimer's disease
20010010912	nrdF
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20030143671	Novel beta-defensins
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20030143600	Detection of extracellular tumor-associated nucleic acid in blood plasma or serum
	using nucleic acid amplification assays
20030143593	Quantative method for measuring gene expression
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20030143241	Hepatitis E virus vaccine and method
20030143219	Nucleic acid molecules encoding a transmembrane serine pro tease 25, the encoded
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20030143202	Anemia
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20030138925	Novel human gene relating to respiratory diseases, obesity, and inflammatory bowel
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20030138854	Method for determining BAGE expression
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20030138772	Method of detecting and/or identifying adeno-associated virus (AAV) sequences and
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SUMMARY OF THE INVENTION

The present invention allows improved detection of such bacterial contaminants, including unconventional forms such as filtering forms (nanoforms), nanobacteria, and L-forms.

Its Principal Applications are the:

• • Detection of very low levels of mycoplasma contamination of cell lines and biological fluids
  • Identification of latent bacterial infections in various pathologies
  • Detection of live forms passing through filters having a pore size of between 100 and 20 nm
  • Direct detection of RNA-containing subunits of bacteria.

Different Primers have been Designed:

• • MOLL primers have been designed initially to detect mollicutes (mycoplasma) species based on the conserved regions of the 16s ribosomal RNA gene. In fact they can also detect gram positive bacteria

1)	Moll Outer Primer (sense)	SEQ ID NO: 1	(AAYGGGTGAGTAACACGT),
2)	Moll Outer Primer (antisense)	SEQ ID NO: 2	(CCCGAGAACGTATTCACCG)
3)	Moll Inner Primer (sense)	SEQ ID NO: 3	(CTACGGGAGGCAGCAGTA)
4)	Moll Inner Primer (antisense)	SEQ ID NO: 4	(GTATCTAATCCTRTTTGCTCCCCA)

• • BACT primers will detect gram positive and gram negative bacteria. The sequence of MOLL primers is included in the degenerated sequence of the BACT primers.

1)	Bact Outer Primer (antisense)	SEQ ID NO: 5	(CCCGRGAACGTATTCACSG),
2)	Bact Inner Primer (sense)	SEQ ID NO: 6	(CTACGGGAGGCWGCAGTRRGGAAT),
3)	Bact Inner Primer (antisense)	SEQ ID NO: 7	(WGGGTATCTAATCCTRTTTGMTCCCCW)

• • The GNEG set of primers is specific of gram negative bacteria. It differs from the Moll 16 out S by a single nucleotide

1)

Gneg Outer Primer (sense)

SEQ ID NO: 8

(RAYGGGTGAGTAAYGYMT),

The present invention therefore provides a method for identifying an RNA form of a bacteria, comprising reverse transcribing RNA material; conducting PCR using primers for a first highly conserved genetic sequence generic of the bacteria; conducting nested PCR using primers for a second highly conserved genetic sequence within the first amplified genetic sequence of the bacteria; and identifying the bacteria based on unconserved amplified sequences linked to the conserved sequences.

It is believed that the Nanoforms are a stable, low metabolic rate form of bacteria, which may be related to pathology, which have characteristic DNA which is generally undetectable by PCR or nested PCR. However, these organisms do have characteristic RNA, and therefore these can be detected by nested RT-PCR. Likewise, because these are now detectable according to the present invention, it is therefore possible to monitor and optimize treatments directed toward clearing these from infected subjects.

It is believed that Nanoforms are involved in human pathology, and further that these low metabolic organisms are involved in a constellation of chronic human diseases. Further, it is believed that some of these Nanoforms may be subcellular, that is, incomplete, and therefore may require association with other Nanoform, or other organisms or cells, for replication or reconstitution as a complete DNA bacterial form. Preliminary evidence suggests that the genetic material within a single Nanoforms is insufficient to reconstitute the entirety of a related bacterial (DNA) form, and therefore that multiple Nanoforms may be required in order to be self-replicating for the complete organism.

For example, multiple Nanoforms may infect a single cell, together constituting a complete genome for the associated DNA bacteria. Reverse transcriptase activity, for example, due to retroviruses, endogenous retroviral sequences, DNA pol I activity, etc., may be sufficiently active to generate the bacterial genome.

These Nanoforms may be biologically associated with retroviruses, such as HIV, which would therefore increase their likelihood of replication, since they would then carry their own reverse transcriptase, and potentially account for replication of sub-cellular fragments. The retroviruses may be passengers within the Nanoforms, and the Nanoforms represent an infectious particle for the virus.

The present invention reveals that the Nanoforms retain conserved sequences of 16S rRNA, and therefore may be targeted on this basis, for example by tetracycline analog antibiotics, especially administered over extended durations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Material and Methods

Oligonucleotide Primers.

The oligonucleotide primers used were:

1)	Moll Outer Primer (sense)	SEQ ID NO: 1	(AAYGGGTGAGTAACACGT),
2)	Gneg Outer Primer (sense)	SEQ ID NO: 8	(RAYGGGTGAGTAAYGYMT),
3)	Bact Outer Primer (antisense)	SEQ ID NO: 5	(CCCGRGAACGTATTCACSG),
4)	Bact Inner Primer (sense)	SEQ ID NO: 6	(CTACGGGAGGCWGCAGTRRGGAAT),
and
5)	Bact Inner Primer (antisense)	SEQ ID NO: 7	(WGGGTATCTAATCCTRTTTGMTCCCCW)

• • where R=G or A, S=G or C, W=A or T, M=A or C, and Y=T or C.

Expected lengths of amplicons are ˜1,200 bp and ˜450 bp with the outer and inner primers, respectively. The primers employed were formulated as equal amounts of each primer within the class identified by the sequence.

Nucleic Acid Preparation and PCR/RT-PCR.

Cell line supernatants (400 μl), human plasma (200-400 μl) and human peripheral blood mononuclear cells (PBMC, 3-10 millions cells) were lyzed with 10 mMTris, pH7.4, 10 mM EDTA, 150 mM NaCl, 0.4% SDS, and 10 μg Proteinase K at 60° C. for 1 h. Nucleic samples were extracted three times with one volume of phenol/chloroform and one time with chloroform and precipitated by addition of 1/10 volume of 3M sodium acetate and two volumes of ethanol at −60° C. for 1 h. Samples were centrifuged 30 min. and the nucleic acid pellets were washed with 70% cold ethanol and solubilized in 10 mM Tris-HCI, pH 8.0. These preparations were stored at −60° C.

PCR reaction mix (50 μl) consisted of 5 mM MgCl2, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 μM dATP, dCTP, dGTP, and DTTP, 0.025% BSA, 1 μM of each outer primer, 1 U Taq polymerase (Roche Molecular biochemicals, Laval, Canada), and 5-10 μl nucleic acid sample. For the first round PCR, the denaturation, annealing, and elongation temperatures and times used were 95° C. for 30 s, 42° C. for 30 s, and 78° C. for 2 m, respectively, for 42 cycles. After the final cycle, the products were kept at 78° C. for 10 m. One μl of the PCR product was subjected to a second round PCR with the set of inner primers. Denaturation, annealing, and elongation temperatures and times used were 95° C. for 30 s, 47° C. for 30 s, and 78° C. for 1 m, respectively, for 42 cycles, followed by a single incubation at 78° C. for 10 m. After the first and second round PCR, 10 μl of PCR product was analyzed by gel electrophoresis using 1.5% agarose, stained with ethidium bromide, visualized under ultraviolet light and photographed. Visible bands with appropriate size were cut and sequenced using the inner primers (DNA Landmarks, St-Jean sur le Richelieu, Canada). Sequence homology search was performed using the BLAST program of the NIH web site.

Samples negative for the appropriate band by PCR were subjected to a first round RT-PCR followed by a second round PCR. RT-PCR reaction mix (50 μl) consisted of 5 mM MgCl2, 50 mM Tris, pH 8.0, 15 mM (NH4)2SO4, 10 mM B-Mercaptoethanol, 500 μM dATP, dCTP, dGTP, and DTTP, 0.025% BSA, 1 μM of each outer primer, and Titan enzyme mix (Roche Molecular biochemicals, Laval, Canada), and 5 μl nucleic acid sample. The reverse transcription step was performed at 42° C. for 30 m. The first and second rounds PCR were performed as described above.

The precautions addressed elsewhere (Kwok and Higuchi, 1989) were followed to minimize the risk of false-positive results caused by the carry-over of previously amplified DNA. For example, extraction of nucleic acids and preparation of PCR/RT-PCR mix were performed under a sterile flow bench, only aliquoted reagents and filter tips were used, and negative controls were incorporated into each run.

Results

All Samples:

First round PCR/RT-PCR: no detection of expected amplicon (˜1,200 bp).

Second round PCR (nested-PCR): all amplicon (˜450 bp) sequences related to bacterial 16S ribosomal RNA gene.

Patients' Lymphocytes (18 Samples):

No 450 bp amplicon detected by nested-PCR from first round PCR.

All samples positive (450 bp) by nested-PCR from RT-PCR.

Therefore, bacteria are in an “RNA state”, and are referred to herein as “Nanoforms”.

Samples of Patients' Plasma:

11 samples/12 positive for 450 bp amplicon by nested-PCR from first round PCR.

Discussion

Cell wall deficient pathogenic microorganisms, which may be mycoplasma, so-called L-forms, or potentially other types, are difficult to detect. Therefore, their involvement in pathology may be vastly under-reported.

It has been found, however, that these organisms have a well-conserved RNA sequences, such as the 16S rRNA, even when corresponding DNA or RNA is undetectable by a traditional polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR) method, which may be detected by nested RT-PCR amplification, using primers according to the present invention.

The present invention therefore provides a sensitive and specific method for detecting bacterial forms, which may be called “Nanoforms”, even when traditional methods fail. This therefore allows diagnosis of pathogens previously unrecognized, and monitoring of treatment thereof.