Real-time polymerase chain reaction for the qualitative and quantitative detection of Mycoplasma gallisepticum

Figures & data

Table 1.  Mycoplasma strains used in M. gallisepticum Q-PCR cross-reactivity testing

Mycoplasma species	Strain ID	Source and history	M. gallisepticum cPCR result	M. gallisepticum Q-PCR result
Mycoplasma gallinaceum	ATCC 33550	ATCC^a strain	–	–
Mycoplasma gallisepticum	ATCC 15302	ATCC strain	+	+
Mycoplasma gallisepticum	Mg F1999^b	AHS field strain, chicken trachea, 1999, Netherlands, M. gallisepticum/chicken//NL/Dev-1608Vin/99	+	+
Mycoplasma gallisepticum	Mg F	S.H.Kleven, chicken trachea, 1975, USA	+	+
Mycoplasma gallisepticum	Mg FS9	G. Czifra, pathogenic challenge strain	+	+
Mycoplasma gallisepticum	Mg R low	S. H. Kleven, pathogenic challenge strain	+	+
Mycoplasma imitans	Mim 4229	ATCC strain 51306—J.M. Bradbury, Liverpool, UK	–	+
Mycoplasma iowae	ATCC 33552	ATCC strain	–	–
Mycoplasma meleagridis	ATCC 25294	ATCC strain	–	–
Mycoplasma meleagridis	Mm F2001	AHS field strain, turkey trachea, 2001, Netherlands	–	–
Mycoplasma synoviae	Ms K1968	S. H. Kleven, turkey, respiratory, 1983, USA	–	–
Mycoplasma synoviae	Ms K870	S. H. Kleven, chicken, respiratory, 1976, USA	–	–
Mycoplasma synoviae	ATCC 25204	WVU 1853	–	–
Mycoplasma synoviae	Ms F1997^c	AHS field strain, chicken trachea, 1997, Netherlands	–	–
Mycoplasma synoviae	Ms F2000^d	AHS field strain, chicken joint, 2000, Netherlands, chicken/NL/DEV/801979rob/00	–	–
^aAmerican Type Culture Collection.
^b M. gallisepticum strain Mg F1999 was isolated from offspring of a breeder flock with serological evidence of infection and clinical signs. Field strains were identified by immunofluorescent staining of colonies on mycoplasma agar and by conventional PCR (IDEXX Laboratories Inc.).
^c M. synoviae strain Ms F1997 was isolated in 1997 from a breeder flock.
^d M. synoviae strain Ms F2000 was isolated from the joint of a chicken (Landman & Feberwee, 2001).

Figure 1. Reproducibility of Q-PCR crossing points of DNA standards compared with log₁₀ M. gallisepticum CFU/ml determined by culture. Mean of five different Q-PCR runs. Bars indicate SEM.

Figure 2. Results of M. gallisepticum DNA standard in Q-PCR and electrophoresis. 1 to 7, 10-fold dilutions from culture A, tested in duplicate, from undiluted (sample 1, 10^6.1 CFU equivalents/ml) to 10⁻⁶ (sample 7, 10^1.1 CFU equivalents/ml); 8, H₂O; M, marker (DNA 50 bp Marker XIII; Roche Applied Science, Mannheim, Germany).

Table 2.  Results of reproducibility of Q-PCR based on M. gallisepticum DNA standards derived from culture A

Culture A^a	Culture^b	Q-PCR^c (mean±SEM)	Tm (°C) (mean±SEM)	Cp (mean±SEM)
M. gallisepticum standard 10^−1	6.1	6.1^c,d±0.02	85.49^d±0.43	17.33^d±1.12
M. gallisepticum standard 10^−2	5.1	5.2±0.02	85.56±0.47	20.10±1.18
M. gallisepticum standard 10^−3	4.1	4.2±0.05	85.53±0.47	23.37±1.50
M. gallisepticum standard 10^−4	3.1	3.0±0.08	85.57±0.47	26.91±1.44
M. gallisepticum standard 10^−5	2.1	1.9±0.08	85.62±0.15	30.56±1.53
M. gallisepticum standard 10^−6	1.1	1.3±0.08	85.74±0.60	32.20±1.97
^a M. gallisepticum DNA standards derived from culture A.
^blog10 M. gallisepticum CFU/ml (culture).
^clog10 M. gallisepticum CFU equivalents/ml calculated from DNA standard curve.
^dMean of five runs.

Table 3.  Results of Q-PCR and culture on time-interval samples taken from culture B

Time (h)	Culture^a	Q-PCR^b
0	0.0	0.0
12	0.0	0.0
48	7.3	6.4
72	7.8	7.4
144	0.0	8.4
^aLog10 M. gallisepticum CFU/ml (culture).
^bLog10 M. gallisepticum CFU equivalents/ml calculated from DNA standard curve.

Figure 3. Effect of dilution of samples on estimation of CFU equivalents/ml using Q-PCR (samples from experiment B). Bar indicates SEM.

Table 4.  Results of in vivo experiments

	Experiment A (n = 3)		Experiment B^a (n = 5)		Experiment C^a (n = 10)
Sampling time (days post-inoculation)	Culture	Q-PCR^b	Culture	Q-PCR^b	Culture	Q-PCR^b
0	0.0^c±0.0	0.0±0.0	0.0^c±0.0	0.0±0.0	0.0^c±0.0	0.0±0.0
2	0.0±0.0	0.0±0.0	–	–	–	–
4	0.0±0.0	1.8±0.40	–	–	–	–
5	–^d	–	7.6±0.65	4.6±0.58	6.9±0.46	5.4±0.28
10	6.4±0.10	5.7±0.20	–	–	–	–
14	–	–	5.9±3.30	4.9±0.51	7.4±0.38	4.9±0.58
15	6.8±0.54	5.5±0.40	–	–	–	–
20	6.9±0.66	4.8±0.97	–	–	–	–
21	–	–	6.3±1.70	3.2±1.43	–	–
25	6.3±0.84	5.1±0.66	–	–	–	–
28	–	–	–	–	1.6±2.22	2.2±1.51
30	3.4±0.26	2.7±0.20	–	–	–	–
35	0.0±0.0	2.4±0.32	–	–	–	–
Data presented as mean±SEM.
^aControl birds in experiments B (n=5) and C (n=5) tested negative in culture and Q-PCR on all sampling days in both experiments.
^bLog10 M. gallisepticum CFU equivalents/ml calculated from DNA standard curve.
^c<50 log10 M. gallisepticum CFU/ml.
^dNo simultaneous Q-PCR testing and culture was carried out.

Landman , W.J.M. and Feberwee , A. 2001 . Field studies on the association between amyloid arthtopathy and Mycoplasma synoviae infection, and experimental reproduction of the condition in brown layers . Avian Pathology , 30 : 629 – 639 .

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