Innovative application of nested PCR for detection of Porphyromonas gingivalis in human highly calcified atherothrombotic plaques

Figures & data

Table 1. Identification of periodontal pathogens in atherothrombotic samples using PCR methods

Authors (year)	Country	Vascular sample	n	Aa (%)	Pg (%)	Tf (%)	Pi (%)
Kurihara et al. (2004) [26]	Japan	Aneurysmal wall	32	0	85	22	31
Marques da Silva et al. (2005) [27]	Norway		56	7.1	0	0	/
Nakano et al. (2009) [28]	Japan		86	30.2	<20	<15	<15
Delbosc et al. (2011) [29]	France		16	/	43.8	/	/
Pyysalo et al. (2013) [30]		Finland	36	<20	<20	/	<20
Haraszthy et al. (2000) [11]	USA	Carotid	50	18	26	30	14
Cairo et al. (2004) [12]	Italy		19	0	0	0	0
Fiehn et al. (2005) [13]¶	Denmark		11	0	0	0	100
Kozarov (2005) [2]	USA		1	100	100	/	/
Aimetti et al. (2007) [14]	Italy		33	0	0	0	0
Figuero et al. (2011) [15]	Spain		42	66.6	78.6	/	/
Rangé et al. (2014) [4]	France		157	0	39	35	33
Fernandes et al. (2014) [16]	Brasil	Carotid, aneurysmal wall	14	/	0	/	7.1
Kozarov et al. (2006) [17]	USA	Carotid, aortic, femoral	9	55.5	88.8	22.2	77.7
Aquino et al. (2011) [18]	Brazil	Carotid, coronary, femoral	30	0	0	/	/
Padilla et al. (2006) [8]	Chile	Carotid, femoral, tibial, popliteal	12	1.7	0	0	0
Toyofuku et al. (2011) [9]	Japan	Carotid, iliac, femoral	53	0	51	0	15
Figuero et al. (2018) [10]^‡	Sweden	Carotid, peripheral, aneurysmal wall	70	2.8	0	2.8	/
Ishihara et al. (2004) [19]	USA	Coronary	51	23.3	21.6	5.9	/
Pucar et al. (2007) [20]	Serbia		15	26.7	53.3	13.3	33.3
Gaetti-Jardim et al. (2009) [21]	Brasil		44	46.2	53.8	25.6	59
Mahendra et al. (2009) [22]	India		51	/	45.1	/	/
Rath et al. (2014) [23]	India		7	42.8	71.4	100	0
Mahalakshmi et al. (2017) [24]	India		65	0	52.3	46.2	32.3
Atarbashi-Moghadam et al. (2018) [25]	Iran		23	17.4	13	/	/
Fiehn et al. (2005) [13]¶	Denmark	Femoral	13	0	7.7	0	100
Okuda et al. (2001) [32]	Japan	Not detailed	26	0	0	0	/
Taylor-Robinson et al. (2002) [33]	UK		32	21.9	0	0	9.4

¶No sample was positive with direct specific PCR; _‡.with nested PCR, Aa: 18.% and Pg: 57.8%

Table 2. Vascular sample preparation for identifying periodontal pathogens

Authors (year)	Transport medium	Storage	Sample homogenization	DNA extraction
Haraszthy et al. (2000) [11]	Sterile saline solution	Processed immediately or frozen at −70°C	Unknown	Instagen® Purification Matrix (Bio rad Laboratories, Hercules, CA)
Okuda et al. (2001) [32]	Unknown	Formalin-fixed, paraffin-embedded blocks	Unknown	Dexpat® (Takara, Otsu, Japan)
Taylor-Robinson et al. (2002) [33]	Unknown	−70°C or −20°C for up to 5 years	Unknown	Flowgen® (Novara House, UK)
Cairo et al. (2004) [12]	Sterile saline solution	−20°C	Addressed with no details	Standard protocol (with proteinase-K and cetyltri-methylammonium bromide)
Ishihara et al. (2004) [19]	Sterilized phosphate-buffered saline and mixed gently	Unknown	Tube pestle with lysis solution	Puregene® kit (Gentra Systems, Minneapolis, Minn.)
Kurihara et al. (2004) [26]	Immediately frozen	−80°C	Addressed with no details	High Pure® PCR Template Preparation Kit (Roche, Mannheim, Germany)
Fiehn et al. (2005) [13]	Reduced transport medium	Unknown	Addressed with no details	Modified SDS extraction method (Sigma-Aldrich, Vallensbaek Strand, Denmark) and DNA clean-up system (purification)
Kozarov (2005) [2]	Pre-reduced tryptic soy broth	Processed immediately	Unknown	DNeasy® Tissue kit (Qiagen Sciences, Valencia, CA)
Marques da Silva et al. (2005) [27]	Pre-reduced anaerobically sterilized Transport medium	−70°C in sterile tubes containing Todd Hewitt broth with 0.5% dimethyl sulfoxide	Sterile mortar with liquid nitrogen under a laminar flow hood	QIAamp® Mini Kit (Qiagen GmbH, Hilden, Germany)
Kozarov et al. (2006) [17]	Pre-reduced transport medium	Unknown	Unknown	QIAmp® Tissue Kit (Qiagen Inc., Valencia, CA)
Padilla et al. (2006) [8]	Saline solution	Cultivated	Addressed with no details	AquaPure® Genomic DNA Isolation kit (BioRad, Hercules, CA, USA)
Aimetti et al. (2007) [14]	Sterile saline solution	−20°C	Mechanical homogeniser (Tissue Laser, QIAGEN, Hilden, Germany)	Phenol-chloroform method and ethanol precipitation
Pucar et al. (2007) [20]	Immediately frozen	−20°C	Unknown	Standard protocol (with proteinase-K)
Elkaïm et al. (2008) [31]	Dry sterile tube immediately frozen with liquid nitrogen	−80°C	Polytron device (Kinematica, Luzern, Switzerland).	Standard protocol (with RNAse A, proteinase K, phenol–chloroform–isoamylic alcohol and EDTA)
Gaetti-Jardim et al. (2009) [21]	Sterile DNA-free saline solution	−20°C	Unknown	Charge Switch® gDNA Mini Tissue kit (Invitrogen)
Nakano et al. (2009) [28]	Sterie saline solution	−20°C	Unknown	Unknown
Mahendra et al. (2009) [22]	Sterilized phosphate buffered saline and mixed gently and then transfer to transport medium	Unknown	Tissue homogenizer (Saiki et al. 1988)	Standard protocol (with lysis solution (Tris, EDTA, Triton), temperature variation and centrifugation)
Aquino et al. (2011) [18]	Sterile microcentrifuge tubes containing trypticase soy broth and dimethyl sulfoxide	−20°C	Unknown	QIAamp® DNA mini kit (Qiagen, Valencia, Spain)
Delbosc et al. (2011) [29]	Unknown	−80°C	Cryopulverized using a freezer mill (Spex Certiprep Ltd)	QIAamp® DNA blood Midi kit (Qiagen) with modifications
Figuero et al. (2011) [15]	Sterile conditions	−20°C	Mechanical homogenizer	G-Nome® DNA kit (MP Biomedicals)
Toyofuku et al. (2011) [9]	Immediately frozen under sterile conditions	−80°C	Addressed with no details	High Pure® PCR Template Preparation Kit (Roche, Mannheim, Germany)
Pyysalo et al. (2013) [30]	Unknown	−70°C	Unknown	Unknown
Fernandes et al. (2014) [16]	Sterile vial containing phosphate-buffered saline	−20°C	Unknown	Standard protocol (with cetyltri-methylammonium bromide)
Figuero et al. (2014) [10]	Sterile conditions	−80°C	Mechanical homogenizer	G-Nome® DNA kit (MP Biomedicals) and purification
Rangé et al. (2014) [4]	Cold Roswell Park Memorial Institute medium (RPMI) (4°C) containing antibiotics plus an antimycotic	Incubated (24 h at 37°C) in a standardized volume (6 mL/g of sample wet weight) of RPMI culture medium supplemented with antibiotics and an antimycotic and TPI at −80°C	Cryopulverized using a freezer mill (Spex Certiprep Ltd)	QIAamp® DNA blood Midi kit (Qiagen) with modifications
Rath et al. (2014) [23]	Saline solution in sterile vial	Frozen in a bath of liquid nitrogen at – 80°C	Addressed with no details	Qiagen® Kit method
Mahalakshmi et al. (2017) [24]	Phosphate buffered saline	−20°C	Unknown	Boiling – lysis method
Atarbashi-Moghadam et al. (2018) [25]	Soaked in saline with sulfate buffer and then placed in Stuart transport medium	Unknown	Addressed with no details	Unknown

Table 3. PCR conditions and identification methods for periodontal pathogens in atherothrombotic samples

Authors (year)	PCR protocol	Cycle (n)	Data analysis
Haraszthy et al. (2000) [11]	Conventional qualitative	30	Hybridization
Okuda et al. (2001) [32]		32 or 36	Clonage and sequencing
Taylor-Robinson et al. (2002) [33]		36	Unknown
Ishihara et al. (2004) [19]		36	Sequencing
Kurihara et al. (2004) [26]		36	Agarose gel
Marques da Silva et al. (2005) [27]		32	Agarose gel
Padilla et al. (2006) [8]		30	Agarose gel
Pucar et al. (2007) [20]		35	Polyacrylamide gel
Elkaïm et al. (2008) [31]		30	Hybridization
Nakano et al. (2009) [28]		36	Unknown
Mahendra et al. (2009) [22]		36	Agarose gel and sequencing
Aquino et al. (2011) [18]		36	Agarose gel
Toyofuku et al. (2011) [9]		36	Gel
Fernandes et al. (2014) [16]		40	Unknown
Rath et al. (2014) [23]		40	Agarose gel
Mahalakshmi et al. (2017) [24]		35 or 36	Gel and sequencing
Atarbashi-Moghadam et al. (2018) [25]		35	Agarose gel and sequencing
Fiehn et al. (2005) [13]	Nested and conventional	30 or 35	Agarose gel and sequencing
Aimetti et al. (2007) [14]	Nested	32	Agarose gel
Figuero et al. (2011) [15]	Nested	35	Agarose gel and sequencing
Figuero et al. (2014) [10]	Nested and quantitative	40	Unknown
Cairo et al. (2004) [12]	Quantitative	35	Agarose gel
Kozarov (2005) [2]		40	Melting curve
Kozarov et al. (2006) [17]		40	Melting curve
Gaetti-Jardim et al. (2009) [21]		40 or 45	Melting curve
Delbosc et al. (2011) [29]		50	Agarose gel
Pyysalo et al. (2013) [30]		60	Melting curve
Rangé et al. (2014) [4]		50	Gel and sequencing

Figure 1. Proposed protocol for Porphyromonas gingivalis 16S rDNA amplification in human calcified atherothrombotic samples in 5 steps. Sterile equipment must be used for all the following steps. (1.) Samples must be transported to the laboratory in a cold (4°C) sterile solution within a few hours after harvesting. (2.) Then, samples must be processed with homogenization. A liquid nitrogen electromagnetic grinding method is preferred over a bead-beating method to maximize nucleic acid preservation. Samples can be stored at −80°C while waiting for homogenization, but freezing and thawing steps should be minimized. (3.) DNA extraction is carried out after pre-incubation with a bacterial lysis buffer using the PrepFiler BTA forensic DNA extraction kit (Life Technologies®) to remove calcifications. (4.) Then, nested PCR is carried out, with conventional PCR as the first step and real-time PCR as the second step (≤40 cycles). (5.) For quality control, the molecular data is obtained using agarose gel migration, melting curve comparison, and sequencing

Figure 2. Images of agarose gels showing the migration of amplicons from an amplification by direct real-time PCR using primers designed for Porphromonas gingivalis. (a) PCR protocol using 40 cycles. (Lane 1:) Negative control. (Lane 2:) Positive control. (Lanes 3–13 and 15, 16:) Samples. (Lane 14:) Ladder. Only the last sample (lane 16) had an amplicon with the same molecular weight as the positive control. (b) PCR protocol using 50 cycles. (Lane 1:) Positive control. (Lanes 2–10 and 12:) Samples. (Lane 11:) Ladder. (Lane 13:) Negative control. All the samples present multiple amplicons

Figure 3. Images of agarose gel showing the migration of amplicons from an amplification by nested PCR (conventional PCR as the first step and real-time PCR as the second step) using primers designed for Porphromonas gingivalis. (Lane 1 and 9:) Ladder. (Lane 2:) Negative control. (Lane 3:) Positive control. (Lanes 4–8 and 10–15:) Samples. Samples from (lanes 4, 7, 11, 13, and 15) had an amplicon with the same molecular weight as the positive control. They were confirmed to be positive by comparison of the melting curves and by sequencing

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