Evaluation of a commercial loop-mediated isothermal amplification assay, 3M^TM Molecular Detection Assay 2 – Campylobacter, for the detection of Campylobacter from poultry matrices

Figures & data

Table 1. Sample preparation and enrichment protocols for Campylobacter spp. detection

		Enrichment medium	Enrichment temperature (± 1°C)	Enrichment time (h)	Enrichment medium	Enrichment temperature (± 2°C)	Enrichment time (h)
Sample matrix	Sample size	MDA2CAM96 (aerobic incubation)^1			MLG 41.04 (microaerophilic incubation)^2
Whole bird carcase and parts rinsed in 400 ml of BPW or nBPW	30 ml of rinsate	30 ml CEB^3	41.5	22–26	30 ml 2X BF-BEB^4	42	46–50
325 g Raw poultry products mixed in 1.625 l of BPW	30 ml of product mixture	30 ml CEB^3	41.5	24–28	30 ml 2X BF-BEB^4	42	46–50
Boots swabs from primary production	1 boot swab	100 ml CEB^4	41.5	22–26

¹MDA2CAM96: 3M™ Molecular Detection Assay 2 – Campylobacter LAMP assay (AOAC PTM, 2018). ²MLG 41.04: BAX® System Real-time PCR assay for Campylobacter spp. (MLG 41.04 2016; MLG 41A.00 2016). ³CEB: Campylobacter enrichment broth.⁴BF-BEB: Blood-free Bolton enrichment broth.

Figure 1. Flow chart for detection of Campylobacter spp. in poultry carcase, parts rinse and raw poultry products by the LAMP method (MDA2CAM96), the MLG 41A PCR reference method (BAX® System Real-time PCR assay for Campylobacter spp.) and the MLG 41.04 culture reference method

Table 2. Detection of Campylobacter spp. in primary production boot swabs using the LAMP method

Boot swab^1	Number of boot swabs^2	Number of positive samples
		MDA2CAM96 Presumptive result^3	Culture confirmed result^4
Natural contamination (uninoculated)	6	0	0
Campylobacter spp.^5	24	23	23

¹Average background aerobic flora was 7.75 log₁₀ CFU/ml. ²Campylobacter spp. presence tested from 30 boot swabs obtained from one farm. Each boot swab was enriched in 100 ml of CEB under aerobic incubation. ³Number of positive samples detected by the LAMP method (MDA2CAM96). ⁴Number of samples detected through culture using the MLG 41.04 culture reference method (MLG 41.04 2016). ⁵Boot swabs (eight for each bacterium, n = 24) were inoculated with each of C. jejuni, C. coli or C. lari at 3 to 7 CFU/boot swab in a bag and incubated aerobically.

Table 3. Comparison between the LAMP method, MDA2CAM96 and the MLG 41A PCR reference method for the detection of Campylobacter spp. in rehang and post-chill carcase nBPW rinsates

Rinsate^1	CEB enrichment (aerobic)			BF-BEB enrichment (microaerophilic)
	Number of rinsates	Presumptive result^2	Culture confirmed result^4	Number of rinsates	Presumptive result^2, 3		Culture confirmed result^4
Rehang carcase		MDA2CAM96			MLG 41A	MDA2CAM96
Natural Contamination (uninoculated)	6	6	6	6	6	6	5
Campylobacter spp.^5	24	24	22	24	22	22	20
Total	30	30	28	30	28	28	25
Post-chill (carcase and parts)		MDA2CAM96			MLG 41A	MDA2CAM96
Whole bird carcase
Natural contamination (uninoculated)	6	4	3	3	1	1	2
Campylobacter spp.^6, 7	9	8	9	12	4	9	10
Parts
Natural contamination (uninoculated)	6	2	1	6	0	0	0
Campylobacter spp.^6, 7	9	7	8	9	3	3	3
Total	30	21	21	30	8	13	15

¹Average background aerobic flora varied from 3.22 log₁₀ CFU/ml for rehang rinsate and 2.05 log₁₀ CFU/ml for post-chill rinsate. ²Number of positive samples detected by the molecular method (LAMP with MDA2CAM96 or PCR with BAX® System Real-time PCR assay for Campylobacter). ³BF-BEB enrichments were also detected with the LAMP assay, MDA2CAM96. ⁴Number of samples detected through culture using the MLG 41.04 culture reference method (MLG 41.04 2016). ⁵Rehang rinsates (eight for each bacterium, n = 24) were inoculated with C. jejuni, C. coli or C. lari at 1 to 5 CFU/rinsate.

⁶Post-chill rinsates (3 carcase and 3 parts for each bacterium, n = 18) were inoculated with C. jejuni, C. coli or C. lari at 1 to 5 CFU/rinsate for the LAMP method.

⁷Post-chill rinsates (6 carcase and 3 parts for C. jejuni and 3 carcase and 3 parts for each of C. coli and C. lari, n = 21) were inoculated at 1 to 5 CFU/rinsate for the MLG 41A PCR reference method.

Table 4. Probability of detection for unpaired comparison between the LAMP method and the MLG 41A PCR reference method for the detection of Campylobacter spp. in poultry matrices

Matrix	Inoculation level	Nr^1	Confirmed positives (reference)	Na^2	Confirmed positives (alternative)	PODr^3	PODa^4	dPOD^5	95% CI^6
Rehang carcase BPW rinse	Natural	6	6	6	6	1	1	0	−0.39	0.39
	Low	24	24	24	24	1	1	0	−0.14	0.14
Rehang carcase nBPW rinse	Natural	6	5	6	6	0.83	1	0.17	−0.25	0.56
	Low	24	20	24	22	0.83	0.92	0.08	−0.12	0.28
Post-chill carcase and parts BPW rinse	Natural	12	10	12	10	0.83	0.83	0	−0.31	0.31
	Low	18	18	18	18	1	1	0	−0.18	0.18
Post-chill carcase and parts nBPW rinse	Natural	9	1	12	4	0.11	0.33	0.22	−0.16	0.51
	Low	21	7	18	15	0.33	0.83	0.50	0.19	0.31
Raw poultry product rinse	Natural	14	10	14	10	0.71	0.71	0	−0.31	0.31
	Low	16	14	16	14	0.87	0.87	0	−0.25	0.25

¹N_r: Total number of samples analysed with the MLG 41A PCR reference method. ²N_a: Total number of samples analysed with the alternative LAMP method. ³POD_r: Probability of detection for the MLG 41A PCR reference method. ⁴POD_a: Probability of detection for the alternative LAMP method. ⁵dPOD: Differential between the POD_a and the POD_r. ⁶95% CI: If the confidence interval of a dPOD does not contain zero, then the difference is statistically significant at the 5% level (Wehling et al. 2011).

AOAC PTM. 2018. “AOAC Performance Tested Method Certificate No. 111803. 3M™ Molecular Detection Assay 2 – Campylobacter.” Accessed June 1 2020. http://members.aoac.org/aoac_prod_imis/AOAC_Docs/RI/19PTM/19C_111803_2MMDA2Camp.pdf.

 Google Scholar

MLG 41.04. 2016. “Isolation and Identification of Campylobacter jejuni/coli/lari from Poultry Rinse, Sponge and Raw Product Samples.” Accessed 1 June 2020. https://www.fsis.usda.gov/wps/wcm/connect/0273bc3d-2363-45b3-befb-1190c25f3c8b/MLG-41.pdf?MOD=AJPERES.

 Google Scholar

MLG 41A.00. 2016. “FSIS Procedure for the Use of a Polymerase Chain Reaction (PCR) Assay for Screening Campylobacter jejuni/coli/lari in Poultry Rinse, Sponge and Raw Product Samples.” Accessed 1 June 2020. https://www.fsis.usda.gov/wps/wcm/connect/ef62eaa8-4d65-4df9-a983-3908fc342b12/MLG-41A.pdf?MOD=AJPERES.

 Google Scholar

Wehling, P., R. A. LaBudde, and M. T. Nelson. 2011. “Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative Methods.” Journal of AOAC International 94: 335–347. doi:10.1093/jaoac/94.1.335.

 PubMed Web of Science ®Google Scholar