Validation of a lateral flow test (MRLAFMQ) for the detection of aflatoxin M₁ at 50 ng l⁻¹ in raw commingled milk

Figures & data

Table 1. Spike milk sample results of MRLAFMQ by means of the ROSA Pearl Reader.

Concentration of aflatoxin M1 (ng l^−1)	Mean (ng l^−1)^a	SD (ng l^−1)	HorRat (CV/PRSDR)^b	Minimum reading	Maximum reading
Blank	2	4	–	0	26
25	25	6	0.80	13	36
50	52	6	0.38	38	68
75	76	8	0.35	54	91

Notes: ^aNumber of replicates used to determine the mean for each concentration: n = 59 for blank, n = 58 for 25 ng aflatoxin M₁ l⁻¹, n = 59 for 50 ng aflatoxin M₁ l⁻¹ and n = 60 for 75 aflatoxin M₁ ng l⁻¹ because of some occasional invalid test developments in the n = 60 replicates.

^bRatio of the coefficient of variation (CV) (= SD/mean) and predicted relative standard deviation (PRSDR) calculated from the Horwitz equation PRSDR (%) = 2C(–0.15), where C is the spike concentration. CV is not calculated for the blank.

Table 2. Spike milk sample results of MRLAFMQ by means of the EZ Reader.

Concentration of aflatoxin M1 (ng l^−1)	Mean (ng l^−1)^a	SD (ng l^−1)	HorRat (CV/PRSDR)^b	Minimum reading	Maximum reading
blank	7	4	–	0	20
25	31	16	1.72	17	91
50	56	10	0.60	39	100
75	81	10	0.41	59	100

Notes: ^aNumber of replicates used to determine the mean for each concentration: n = 59 for blank, n = 58 for 25 ng aflatoxin M₁ l⁻¹, n = 60 EZ for 50 ng aflatoxin M₁ l⁻¹ and n = 60 for 75 aflatoxin M₁ ng l⁻¹ because of some occasional invalid test developments in the n = 60 replicates.

^bRatio of the coefficient of variation (CV) (= SD/mean) and predicted relative standard deviation (PRSDR) calculated from the Horwitz equation PRSDR (%) = 2C(–0.15), where C is the spike concentration. CV is not calculated for the blank.

Table 3. Test selectivity of MRLAFMQ test for aflatoxin M₁.

Chemical compound (concentration in ng l^−1)	EZ Reader		ROSA Pearl Reader
	Mean (ng l^−1)	Result	Mean (ng l^−1)	Result
Benzylpenicillin (40)	2	Negative	0	Negative
Cefalonium (200)	5	Negative	5	Negative
Oxytetracycline (1,000)	0	Negative	0	Negative
Erythromycin (400)	0	Negative	2	Negative
Neomycin (15,000)	2	Negative	5	Negative
Enrofloxacin (1,000)	0	Negative	0	Negative
Sulfadiazine (1,000)	4	Negative	4	Negative
Trimethoprim (500)	0	Negative	1	Negative
Dapson (50)	5	Negative	0	Negative
Ochratoxin A (500)	4	Negative	1	Negative
Zearalenone (250)	0	Negative	5	Negative
Deoxynivalenol (500)	3	Negative	5	Negative
Fumonisin B1 (500)	4	Negative	1	Negative
Aflatoxin M2 (100)	22	Negative	17	Negative
Aflatoxin M2 (200)	19	Negative	22	Negative
Aflatoxin M2 (300)	27	Negative	27	Negative
Aflatoxin M2 (400)	24	Negative	26	Negative
Aflatoxin M2 (500)	37	Negative	39	Negative/pos
Aflatoxin M2 (600)	43	Negative/positive	41	Negative/positive

Note: Data are the mean (ng l⁻¹) of n = 2 tests and the interpretation is based on a 40 ng l⁻¹ cut-off for each reader type.

Table 4. Reader repeatability of MRLAFMQ.

Concentration of aflatoxin M1 (ng l^−1)	ROSA Pearl Reader, SD of the square of pair differences	EZ Reader, SD of the square of pair differences
Blank	0.50	0.71
25	0.88	1.72
75	0.77	1.77

Table 5. Test repeatability of MRLAFMQ.

Concentration of aflatoxin M1 (ng l^−1)	ROSA Pearl Reader, SD of the square of pair differences	EZ Reader, SD of the square of pair differences
blank	1.72	2.83
25	4.53	4.06
75	6.11	7.00

Table 6. Summary of negative and positive results with different farm milk, tanker/truck milk and reconstituted milk powder samples.

	Aflatoxin M1 (ng l^−1)
	ROSA Pearl Reader	EZ Reader
Number of farm milk samples	131	126^a
Number of initial positive	0	0^a
Number confirmed positive	0	0
Number of tanker/truck milk samples	123	123
Number of initial positive	1	0
Number confirmed positive	0	0
Number powder milk samples	20	20
Number of initial positive	0	0
Number confirmed positive	0	0
Total initial positives	1	0^a
Total confirmed positives	0	0
Total initial negative results	273	269^a
Total confirmed negatives	274	269^a
Mean ± SD of farm tanks	9 ± 7	16 ± 12
Minimum reading	0	0
Maximum reading	34	34^a
Mean ± SD of truck tanks	10 ± 7	12 ± 5
Minimum reading	0	1
Maximum reading	41	31

Note: ^aFive farmer samples are not reported because they tested positive due to mis-insertion into the EZ Reader. They immediately tested negative on strip reinsertion into the reader.

Table 7. Effect of change in milk volume on MRLAFMQ readings.

	ROSA Pearl Reader			EZ Reader
	Milk volume			Milk volume
Sample	300 μl	270 μl	330 μl	300 μl	270 μl	330 μl
Blank milk
Mean	8	5	4	12	9	10
Minimum	3	0	2	8	6	9
Maximum	15	8	8	14	16	10
Aflatoxin M1 25 ng l^−1
Mean	24	27	20	29	29	30
Minimum	22	19	15	26	24	26
Maximum	26	34	23	32	31	82
Aflatoxin M1 50 ng l^−1
Mean	52	48	53	55	51	61
Minimum	47	39	51	53	49	54
Maximum	59	52	56	58	54	67

Table 8. Effect of the milk temperature on MRLAFMQ readings.

	ROSA Pearl Reader				EZ Reader
	Milk temperature				Milk temperature
	3°C	10°C	15°C	20°C	3°C	10°C	15°C	20°C
Blank milk
Mean	1	0	0	2	6	4	6	6
Minimum	0	0	0	0	4	1	3	5
Maximum	3	1	0	5	9	9	9	7
Aflatoxin M1 25 ng l^−1
Mean	27	23	25	24	29	30	31	34
Minimum	24	21	24	19	26	27	30	31
Maximum	31	24	26	27	31	32	34	38
Aflatoxin M1 50 ng l^−1
Mean	66	55	46	51	57	64	60	61
Minimum	62	53	37	46	51	62	55	54
Maximum	72	57	54	61	62	66	63	68

Figure 1. Effect of milk composition or quality effects on the screening of blank milk using MRLAFMQ and EZ Reader. Maximum reading (▾), average reading (■), minimum reading (), control point (40 ng l⁻¹) dividing positive from negative (▬▬); 1 = reference: normal raw cows’ milk, 2 = somatic cell count > 10⁶ ml⁻¹, 3 = high bacterial count (>5 × 10⁵ ml⁻¹), 4 = low fat content (<2 g 100 ml⁻¹), 5 = high fat content (>6 g 100 ml⁻¹), 6 = low protein (<3 g 100 ml⁻¹), 7 = high protein (>4 g 100 ml⁻¹), 8 = low pH (6.0), 9 = high pH (7.5).

Figure 2. Effect of milk composition or quality on the detection of 50 ng l^–1 aflatoxin M₁ in milk using MRLAFMQ and EZ Reader. Maximum reading (▾), average reading (■), minimum reading (), control point (40 ng l⁻¹) dividing positive from negative (▬▬); 1 = reference: normal raw cows’ milk, 2 = somatic cell count > 10⁶ ml⁻¹, 3 = high bacterial count (>5 × 10⁵ ml⁻¹), 4 = low fat content (<2 g 100 ml⁻¹), 5 = high fat content (>6 g 100 ml⁻¹), 6 = low protein (<3 g 100 ml⁻¹), 7 = high protein (>4 g 100 ml⁻¹), 8 = low pH (6.0), 9 = high pH (7.5).

Table 9. Pasteurised whole-milk spike sample results of MRLAFMQ by means of the ROSA Pearl Reader.

Concentration of aflatoxin M1 (ng l^−1)	Mean (ng l^−1)	SD (ng l^−1)	Minimum reading	Maximum reading
Blank	11	6	0	21
25	31	5	15	42
50	56	6	45	66
75	77	8	59	95

Table 10. Pasteurised whole-milk spike sample results of MRLAFMQ by means of the EZ Reader.

Concentration of aflatoxin M1 (ng l^−1)	Mean N = 60 (ng l^−1)	SD (ng l^−1)	Minimum reading	Maximum reading
Blank	11	6	0	24
25	33	6	21	45
50	58	8	42	79
75	78	7	63	96

Figure 3. Screening of different blank milk types using MRLAFMQ and ROSA Pearl Reader. Maximum reading (▾), average reading (■), minimum reading (), control point dividing positive from negative (▬▬) ; 1 = reference: normal raw cows’ milk, 2 = UHT milk, 3 = sterilised milk, 4 = reconstituted powder, 5 = frozen–thawed, 6 = goats’ milk, 7 = mares’ milk.

Figure 4. Detection of 50 ng l⁻¹ aflatoxin M₁ in different milk types using MRLAFMQ and ROSA Pearl Reader. Maximum reading (▾), average reading (■), minimum reading (), control point dividing positive from negative (▬▬) ; 1 = reference: normal raw cows’ milk, 2 = UHT milk, 3 = sterilised milk, 4 = reconstituted powder, 5 = frozen–thawed, 6 = goats’ milk, 7 = mares’ milk.

Table 11. Testing of blank milk, milk spiked with 25 ng l⁻¹ aflatoxin M₁, 50 ng l⁻¹ aflatoxin M₁ and 75 ng l⁻¹ aflatoxin M₁ using two different lots of MRLAFMQ.

	ROSA Pearl Reader						EZ Reader
	Lot 010			Lot 011			Lot 010			Lot 011
Sample	Mean	Minimum	Maximum	Mean	Minimum	Maximum	Mean	Minimum	Maximum	Mean	Minimum	Maximum
Blank milk	2	0	9	1	0	7	7	0	43	3	0	40
25 ng l^−1 Aflatoxin M1	30	18	40	24	16	34	32	23	43	26	17	35
50 ng l^−1 Aflatoxin M1	56	40	89	55	41	81	60	40	77	58	41	74
75 ng l^−1 Aflatoxin M1	77	49	95	74	38	100	82	59	100	77	44	98

Figure 5. Results for daily blank samples () and Charm-positive control (▾) by means of the EZ Reader.

Figure 6. Results for daily blank samples () and 50 ng l⁻¹ aflatoxin M₁ spiked standard (▾) by means of the EZ Reader.