Real-time PCR as a Diagnostic Tool for Bacterial Diseases

Max Maurin

Expert Rev Mol Diagn. 2012;12(7):731-754.

Abstract and Introduction

Abstract

In recent years, quantitative real-time PCR tests have been extensively developed in clinical microbiology laboratories for routine diagnosis of infectious diseases, particularly bacterial diseases. This molecular tool is well-suited for the rapid detection of bacteria directly in clinical specimens, allowing early, sensitive and specific laboratory confirmation of related diseases. It is particularly suitable for the diagnosis of infections caused by fastidious growth species, and the number of these pathogens has increased recently. This method also allows a rapid assessment of the presence of antibiotic resistance genes or gene mutations. Although this genetic approach is not always predictive of phenotypic resistances, in specific situations it may help to optimize the therapeutic management of patients. Finally, an approach combining the detection of pathogens, their mechanisms of antibiotic resistance, their virulence factors and bacterial load in clinical samples could lead to profound changes in the care of these infected patients.

Introduction

This review will focus on recent advances in the development of quantitative real-time PCR (qPCR)-based diagnostic tools allowing detection and optional quantification of bacterial DNA in clinical specimens. The authors will describe the existing and potential contributions of qPCR tests for diagnosis, prognosis and treatment of diseases caused by specific bacterial species, including assessment of the antibiotic susceptibilities of these pathogens. Previous reviews have already addressed these issues.^[1–3] The author's goal is to describe the latest advances in this area, citing the most recent and relevant publications, without attempting to be exhaustive. Only qPCR tests applied to human specimens will be studied. The article aims to highlight the many possibilities offered by these new techniques, and also the low number of solutions marketed as compared with potential applications, as evidenced by the large number of in-house tests developed in this area (Table 1).

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Next Section

Abstract and Introduction
Real-time PCR Technology
Diagnostic Applications for Bacterial Diseases
qPCR for Detection of Antibiotic Resistances
Quantification of Bacterial Load Using qPCR
Expert Commentary
Five-year View
References
Sidebar

Table 1. Quantitative real-time PCR tests developed for specific pathogenic bacteria species. For each test the qPCR technology used is specified.
Pathogen	qPCR test (qPCR instrument)	Target gene (protein)	Probe type	Refs.
Staphylococcus aureus/methicillin-resistant S. aureus	In-house (LightCycler)	sa442, nuc (nuclease), mecA (PLP2a)	Dual-Hyb	[23]
	Roche Light Cycler MRSA (LightCycler)	mecA	Dual-Hyb	[24]
	In-house (LightCycler 2.0)	mecA, nuc	Dual-Hyb	[21]
Methicillin-resistant S. aureus carriage	BD GeneOhm™ MRSA ACP (SmartCycler® II)	mecA	Beacon	[16]
	BD GeneOhm MRSA/Xpert® MRSA (SmartCycler II/GeneXpert®)	mecA and chromosomal gene/SPA, SCCmec and mecA	Beacon	[17]
	Xpert MRSA (GeneXpert)	SPA, SCCmec, mecA	Beacon	[18]
	LightCycler MRSA/BD GeneOhm MRSA	mecA	Dual-Hyb	[19]
	BD GeneOhm MRSA	SPA, SCCmec, mecA	Beacon	[20]
Streptococcus pneumoniae	In-house (ABI Prism® 7000)	lytA (autolysine)	TaqMan	[26]
	In-house (ABI Prism 7300)	ply (pneumolysin)	TaqMan	[27]
	In-house (Rotor-Gene 3000 and LightCycler)	ply, lytA, Spn9802 intergenic region	TaqMan and Dual-Hyb	[29]
	In-house (LightCycler)	Ply, lytA	Dual-Hyb	[30]
	In-house (Stratagene M×3000P™, ABI Prism 7500)	lytA, ply, psaA (Mn ABC transporter substrate-binding lipoprotein)	TaqMan	[31]
Streptococcus agalactiae	Cepheid, Xpert GBS (GeneXpert)	cfb (diffusible extracellular protein)	Beacon	[33,37]
	BD Max™ GBS (BD Max)	Cfb	Scorpion	[34]
	Somagen®, IDI StrepB™ (SmartCycler)	Cfb	NS	[35]
	In-house (SmartCycler)	ssrA (tmRNA)	Dual-Hyb	[36]
Streptococcus pyogenes	In-house (Mx3005P™, Stratagene)	NS	TaqMan	[40]
Streptococcus pyogenes	Roche Applied Science (LightCycler)	pts1 (phosphotransferase)	Dual-Hyb	[39]
Neisseria meningitidis	In-house LAMP (ABI Prism 7000)	ctrA (outer membrane protein regulating capsule transport)	SYBR Green	[43]
	In-house (NS)	ctrA	TaqMan	[47]
	In-house (LightCycler 480)	ctrA	Taqman	[48]
	In-house (ABI Prism 7000)	ctrA, siadD (sialic acids of the polysaccharide capsule)	TaqMan	[44,45]
Haemophilus influenzae	In-house (NS)	hpd (protein D)	TaqMan	[50]
	In-house (Rotor-Gene® 3000)	ompP6, rnpB (RNase P RNA), bexA (capsule)	TaqMan	[51]
	In-house (ABI Prism 7500)	bexA, cap locus (capsular types a to f)	TaqMan	[52]
Shiga toxin-producing Escherichia coli	In-house (ABI Prism 7000 or 7500)	stx1 and stx2 (shiga-like toxins)	TaqMan, SYBR Green	[55,56]
	In-house (LightCycler)	stx1 and stx2	Dual-Hyb	[58]
	In-house (Rotor-Gene 6000)	stx1 and stx2	SYBR Green	[59]
	In-house (PTC200 and Chromo 4™ optical detector)	stx1 and stx2	SYBR Green	[60]
Mycobacterium tuberculosis complex	Xpert MTB/RIF (GeneXpert)	rpoB (RNA polymerase subunit B)	Beacon	[67,68]
M. tuberculosis complex	Xpert MTB RIF/Roche Cobas® TaqMan MTB	rpoB/16S rRNA gene	Beacon/TaqMan	[70]
	Xpert MTB RIF/in-house IS6110 qPCR	rpoB/IS6110	Beacon/TaqMan	[69]
	Roche Cobas TaqMan MTB (TaqMan 48)	16S rRNA gene	TaqMan	[71]
	In-house (Stratagene M×3005P)	IS6110	TaqMan	[73]
	In-house (SmartCycler II)	IS6110	SYBR Green	[74]
Non-tuberculous mycobacteria	In-house, Mycobacterium avium subsp. paratuberculosis (ABI 7500)	IS900, F57	TaqMan	[63]
	In-house, M. avium subsp. paratuberculosis	MAP2765c, MAP0865, IS900,	TaqMan	[64]
	In-house, M. avium subsp. hominissuis (LightCycler 480)	IS901, IS1245	TaqMan	[65]
	In-house, six species	hsp65 (heat shock protein 65)	Dual-Hyb	[66]
Chlamydia trachomatis and Neisseria gonorrhoeae	Siemens VERSANT® CT/GC (kPCR system)	Cryptic plasmid and pivNG	NS	[77]
	Abbott RealTime™ CT/NG (m2000)	Cryptic plasmid and Opa gene	TaqMan	[78,80,81]
	Cobas 4800 CT/NG (Cobas 4800)	Cryptic plasmid, chromosomal gene and DR9	NS	[79]
Chlamydophila pneumoniae	In-house LUX-PCR or TaqMan-PCR (NS)	pm4 (polymorphic membrane protein 4), ompA (major outer membrane protein A)	LUX or TaqMan	[93]
	In-house (ABI Prism 7700)	ompB (major outer membrane protein B)	TaqMan	[90]
	In-house (SmartCycler)	ompA (major outer membrane protein A), 16S rRNA gene	TaqMan	[88]
	In-house (ABI Prism 7700)	53-kDa protein gene	TaqMan	[95]
	Nuclisens®, bioMérieux (real-time NASBA)	16S rRNA gene	Beacon	[86]
Chlamydophila psittaci	Takara Bio Dice™ Real-Time System TP800	envB (cysteine-rich protein)	SYBR Green	[92]
	In-house (Corbett 3000)	16S rRNA gene, 16–23S rRNA interspacer	SYBR Green	[87]
	In-house (LightCycler)	ompA (outer membrane protein A)	TaqMan	[89]
	In-house (ABI Prism 7000)	incA (inclusion membrane protein)	TaqMan	[91]
Rickettsia	In-house (ABI Prism 7900)	16S rRNA gene, gltA (citrate synthase)	TaqMan	[96]
	In-house (LightCycler)	RC0338, RP278, RAF-pORF1267, RC0743, 23S rRNA gene, intergenic sequence	TaqMan	[97]
	In-house (SmartCycler II)	17-kDa antigen gene, ompB	TaqMan	[99]
	In-house (Bio-Rad™ iQ5)	ompA, 17-kDa lipoprotein precursor gene, 56-kDa major outer membrane protein gene	TaqMan	[98]
Coxiella burnetii	In-house (MX-4000)	IS1111	NS	[101]
	In-house (ABI Prism 7500)	IS1111	TaqMan	[102]
	In-house (LightCycler)	IS1111, IS30a	TaqMan	[100]
Anaplasma phagocytophilum	In-house LAMP (Teramecs LA200)	msp2 (major surface protein 2)	TaqMan	[105]
Anaplasma phagocytophilum	In-house (iCycler™)	msp2	TaqMan	[106]
Ehrlichia chaffeensis	In-house (iCycler)	Dsb (disulfide bond formation protein)	SYBR Green	[107]
Bartonella	In-house (NS)	NADH dehydrogenase g-subunit gene	None	[110]
	In-house (ABI Prism 7900 HT)	groEL (heat shock protein)	Taqman	[111]
	In-house (LightCycler)	pap31 (extracellular matrix adhesion)	SYBR Green	[112]
	In-house (LightCycler)	gltA (citrate synthase)	Dual-Hyb	[113]
	In-house (LightCycler)	ribC (riboflavin synthase)	SYBR Green	[114]
Mycoplasma pneumoniae	In-house (LightCycler)	RepMP2 (repetitive element of the P1 cytadhesin gene)	Dual-Hyb	[116]
	In-house (Microfluidic)	Mp181 (toxin)	Taqman	[117]
	In-house HRM-qPCR (Rotor-Gene 6000)	RepMP2/3 regions within the P1 gene	SYBR Green	[122]
	Five commercial kits	P1 cytadhesin gene	TaqMan	[118]
	In-house (ABI 7500)	Mp3 and Mp7 (ATPases), Mp181 (CARDS toxin)	TaqMan	[119, 120]
	In-house (LightCycler)	repMp1 (repetitive element of the P1 cytadhesin gene)	TaqMan	[121]
Mycoplasma hominis	In-house (LightCycler 480)	yidC (membrane protein translocase)	TaqMan	[124]
Mycoplasma hominis	In-house (ABI Prism 7900)	16S rRNA gene	TaqMan	[125]
Mycoplasma genitalium	In-house (Rotor-Gene 6000)	pdhD (dihydrolipoamide dehydrogenase)	TaqMan	[129]
	In-house (LightCycler 480)	MgPa (adhesion protein), 16S rRNA gene	TaqMan	[126]
	In-house (Rotor-Gene 6000)	mg219	TaqMan	[130]
Ureaplasma urealyticum and Ureaplasma parvum	In-house (Bio-Rad iQ5)	urease B gene	TaqMan	[127]
Ureaplasma urealyticum and Ureaplasma parvum	In-house (LightCycler 2.0)	UUR10_0680 and UP063	Dual-Hyb	[128]
Helicobacter pylori	In-house (Rotor-Gene 6000)	16S rRNA and 23S rRNA genes	TaqMan	[134]
	In-house (Light Cycler)/Seeplex® ClaR-H. pylori ACE	23S rRNA gene	Dual-Hyb/none	[135]
	Ingenetix ClariRes (LightCycler)	23S rRNA gene	SYBR Green	[136]
	In-house(Rotor-Gene 6000)	ureC (urease α subunit)	TaqMan	[133]
Clostridium difficile	Xpert CD/Epi (GeneXpert)/Meridian Illumigene™ CD (LAMP)	tcdB, cdt, tcdC deletion of 027 strains/tcdA	Beacon	[138]
	In-house (ABI Prism 7300)	tcdB, tcdC deletion of 027 strains	TaqMan	[139]
	In-house (LightCycler 2.0)	tcdA, tcdB	TaqMan	[140]
	In-house (ABI Prism 7500)/BD GeneOhm CD (SmartCycler)	tcdA, tcdB, tcdC deletion of 027 strains/tcdB	TaqMan	[141]
	In-house (ABI Prism 7500)	tcdA, tcdB	TaqMan	[142]
	BD GeneOhm CD (SmartCycler)	tcdB	TaqMan	[143]
Legionella	In-house, Sg1-qPCR (Bio-Rad Chromo4 system)	wzm and wzt (lipopolysaccharide biosynthesis proteins)	TaqMan	[146]
	In-house (LightCycler)	16S rRNA gene, MIP	Dual-Hyb	[147]
	In-house (ABI Prism 7900)	23S-5S rRNA intergenic spacer region	TaqMan	[148]
	In-house (ABI Prism 7900)	16S rRNA gene, MIP	TaqMan	[149]
Bordetella pertussis and Bordetella parapertussis	In-house (ABI 7500)	IS481, IS1001, ptxS1 (pertussis toxin subunit S1)	TaqMan	[152]
	In-house (SmartCycler) and four commercial tests	IS481	TaqMan	[153]
	In-house (variable)	IS481	Dual-Hyb, TaqMan, Beacon	[154]
	In-house (LightCycler)	ptxA-pr (pertussis toxin S1 promoter), IS481	Dual-Hyb	[155]
	In-house (LightCycler)	BP283 and BO485 DNA regions	TaqMan	[156]
	In-house (ABI 7900, ABI 7500, SmartCycler II, Rotor-Gene 3000)	IS481, IS1001	TaqMan	[157]
Corynebacterium diphtheria and Corynebacterium ulcerans	In-house (LightCycler)	tox (diphtheria toxin)	Dual-Hyb	[160]
Corynebacterium diphtheria and Corynebacterium ulcerans	In-house (Stratagene Mx3000P)	tox (diphtheria toxin)	TaqMan	[162]
Brucella	In-house (LightCycler)	IS711	TaqMan	[165]
	In-house (LightCycler)	BCSP31 gene	TaqMan	[166]
	In-house (LightCycler)	BCSP31 gene	TaqMan	[167]
	In-house (LightCycler 2.0)	BCSP31 gene	Dual-Hyb	[168]
Francisella tularensis	In-house (LightCycler 2.0)	ISFtu2, Tul4 (surface protein gene)	TaqMan	[169,170]
	In-house (SmartCycler II)	FTT0376, FTT0523	TaqMan	[170]
	In-house (LightCycler 1.2)	pdpD (pathogenicity determinant protein D), AY06 (junction ISFtu2 and flanking 3′ DNA)	Taq Man	[171]
Leptospira	In-house (Bio-Rad CFX96)	secY (preprotein translocase), lfb1 (protein of unknown function), lipL32 (lipoprotein)	SYBR Green and TaqMan	[173]
	In-house (Rotor-Gene 3000)	rrs (16SrRNA gene), lipL32 (outer membrane lipoprotein)	TaqMan	[174]
	In-house (Bio-Rad iQ5)	secY (preprotein translocase)	SYBR Green	[175]
	In-house (ABI 7500)	lipL32 (outer membrane lipoprotein)	TaqMan	[176]
	In-house LAMP (MJ Opticon 2)	lipL41 (outer membrane protein)	SYBR Green	[177]
Treponema pallidum	In-house (LightCycler 2.0)	tpp47 (47-kDa membrane lipoprotein)	SYBR Green	[178]
Treponema pallidum	In-house (Rotor-Gene 3000)	polA (DNA polymerase)	TaqMan	[180]
Borrelia (Lyme disease)	In-house (LightCycler)	hbb (histone-like protein)	Dual-Hyb	[185]
	In-house (LightCycler 2.0)	borrelial plasminogen binding protein gene	Dual-Hyb	[184]
	In-house (LightCycler 2.0)	ospA (outer surface protein A)	TaqMan	[183]
Bacillus anthracis	In-house (ABI 7900)	capA, capB, capC (capsule), lef and pag (toxins)	Beacon	[187]
Bacillus anthracis	In-house (ABI 7000 SDS or 7700 SDS)	pXO1 (protective antigen)	TaqMan	[188]
Yersinia pestis	In-house (Rotor-Gene 3000)	pla (plasminogen activator), caf1 (F1 antigen), ymt (phospholipase D)	TaqMan	[190]
Yersinia pestis	In-house (Rotor-Gene 2000)	ypo2088 (putative methyltransferase), pla (plasminogen activator)	TaqMan	[191]
Enterococcus (VRE)	BD GeneOhmVanR (SmartCycler II)	vanA, vanB	TaqMan	[227]
	Xpert VanA/vanB, Cepheid (GeneXpert)	vanA, vanB	Beacon	[228]
	Roche Light Cycler VRE (LightCycler)	vanA, vanB	Dual-Hyb	[229]
	BD GeneOhm VanR (SmartCycler II)	vanA, vanB	TaqMan	[230]

Probes: TaqMan (5' nuclease probe), Dual-Hyb (dual-hybridization probe), Beacon (molecular beacon), Scorpion (bi-labeled fluorescent probe/primer hybrids), SYBR green (fluorescent DNA intercalating agent).
CARDS: Community-acquired respiratory distress syndrome; HRM-qPCR: qPCR with high resolution melt analysis; LAMP: Loop-mediated isothermal amplification; LUX: Light upon extension PCR; MIP: Macrophage infectivity potentiator; NS: Not specified; VRE: Vancomycin-resistant Enterococci.