Comparison of the Forensic Testing Performance of Six STR Kits

doi:10.12116/j.issn.1004-5619.2024.540105

Abstract

Abstract:

Objective To compare the technical performance indicators of six commonly used STR kits, and to evaluate their capabilities in forensic testing. Methods The STR genotyping results of forensic DNA standard sample 9948 with different template quantities were analyzed using Identifiler^TM Plus, AGCU EX28CS, NHID^® 25A, Verifiler^TM Plus, VersaPlex^TM 27PY, and FastDirect^TM 25A. Differen-ces in detection sensitivity, allelic drop-out/in, allele peak height and heterozygote balance were eva-luated. Various PCR inhibitors, including humic acid, heme, indigo, calcium ions and ethylenediaminetetraacetic acid （EDTA）， were added to the multiplex amplification systems to compare the maximum tolerance levels of the six kits to PCR inhibitors. Results The detection sensitivity of Identifiler^TM Plus, AGCU EX28CS and VersaPlex^TM 27PY was as low as 100 pg, whereas the sensitivity of FastDirect^TM 25A, NHID^® 25A and Verifiler^TM Plus reached 50 pg. At different template quantities, the number of STR loci detected by the five kits was greater than that of Identifier^TM Plus. When detecting templates of 50 pg or less, the total number of allelic drop-out/in observed with the Identifiler^TM Plus was lower than that observed with the other five kits. The allele peak heights detected by AGCU EX28CS at 200 and 100 pg template quantities and by VersaPlex^TM 27 PY at 50 pg were significantly lower than those detected by Identifiler^TM Plus （P<0.05）. Regarding resistance to inhibitors, NHID^® 25A showed stronger tolerance to humic acid （≤250 ng/μL）, heme （≤0.6 mmol/L） and calcium ions （≤1.4 mmol/L） than other kits. It was found that VersaPlex^TM 27PY showed the strongest tolerance to indigo （≤0.048 mol/L）; and FastDirect^TM 25A showed the strongest tolerance to EDTA （≤1.0 mmol/L）. For heme, AGCU EX28CS and FastDirect^TM 25A showed lower tolerance than Identifiler^TM Plus, whereas the other three kits exhibited higher tolerance. For other inhibitors, all five kits demonstrated tolerance comparable to or higher than that of Identifiler^TM Plus. Conclusion Compared with Identifiler^TM Plus, AGCU EX28CS, FastDirect^TM 25A, NHID^® 25A, VersaPlex^TM 27PY and Verifiler^TM Plus show no obvious overall disadvantage in detection sensitivity, allelic drop-out/in, allele peak height, heterozygote balance and inhibitor resistance and even demonstrate superior performance in certain technical indicators.

Key words: forensic genetics, short tandem repeat, sensitivity, heterozygote balance, PCR inhibitor, testing kits

CLC Number:

Yi-hua SUN, Jian CHEN, Yong-sheng LI. Comparison of the Forensic Testing Performance of Six STR Kits[J]. Journal of Forensic Medicine, 2026, 42(1): 26-33.

Figures/Tables 6

References 25

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试剂盒	预变性	热循环	终延伸	保温/℃	扩增时间/min
Identifiler^TM Plus	95 ℃ 11 min	94 ℃ 20 s，59 ℃ 3 min；29次	60 ℃ 10 min	4	152
AGCU EX28CS	95 ℃ 5 min	94 ℃ 10 s，60 ℃ 1 min；29次	60 ℃ 7 min	4	93
FastDirect^TM 25A	95 ℃ 5 min	94 ℃ 10 s，59 ℃ 80 s；30次	60 ℃ 10 min	15	96
NHID^® 25A	95 ℃ 1 min	95 ℃ 10 s，59 ℃ 1 min，72 ℃ 20 s；29次	60 ℃ 10 min	4	88
VersaPlex^TM 27PY	96 ℃ 5 min	96 ℃ 5 s，60 ℃ 1 min；29次	60 ℃ 10 min	4	82
Verifiler^TM Plus	96 ℃ 1 min	第一步：96 ℃ 10 s，62 ℃ 90 s；2次第二步：96 ℃ 10 s，59 ℃ 90 s；27次	60 ℃ 5 min	4	77

试剂盒	预变性	热循环	终延伸	保温/℃	扩增时间/min
Identifiler^TM Plus	95 ℃ 11 min	94 ℃ 20 s，59 ℃ 3 min；29次	60 ℃ 10 min	4	152
AGCU EX28CS	95 ℃ 5 min	94 ℃ 10 s，60 ℃ 1 min；29次	60 ℃ 7 min	4	93
FastDirect^TM 25A	95 ℃ 5 min	94 ℃ 10 s，59 ℃ 80 s；30次	60 ℃ 10 min	15	96
NHID^® 25A	95 ℃ 1 min	95 ℃ 10 s，59 ℃ 1 min，72 ℃ 20 s；29次	60 ℃ 10 min	4	88
VersaPlex^TM 27PY	96 ℃ 5 min	96 ℃ 5 s，60 ℃ 1 min；29次	60 ℃ 10 min	4	82
Verifiler^TM Plus	96 ℃ 1 min	第一步：96 ℃ 10 s，62 ℃ 90 s；2次第二步：96 ℃ 10 s，59 ℃ 90 s；27次	60 ℃ 5 min	4	77

模板质量/pg	试剂盒	等位基因基因座数/个		基因座		常染色体STR
模板质量/pg	试剂盒	缺失	插入	平均检出数/个	平均检出率/%	平均检出数/个	平均检出率/%
50	Identifiler^TM Plus	4	0	14.67±0.47	91.67±2.95	13.33±0.47	88.89±3.14
	AGCU EX28CS	6	2	26.00±1.41	92.86±5.05	21.33±1.25	92.75±5.42
	FastDirect^TM 25A	0	0	25.00±0.00	100.00±0.00	23.00±0.00	100.00±0.00
	NHID^® 25A	0	0	25.00±0.00	100.00±0.00	23.00±0.00	100.00±0.00
	VersaPlex^TM 27PY	2	0	26.33±0.47	97.53±1.75	22.33±0.47	97.10±2.05
	Verifiler^TM Plus	0	0	25.00±0.00	100.00±0.00	23.00±0.00	100.00±0.00
25	Identifiler^TM Plus	14	4	11.00±2.16	68.75±13.50	10.33±1.70	68.59±11.33
	AGCU EX28CS	25	1	19.67±2.87	70.24±10.24	15.67±0.47	68.12±2.05
	FastDirect^TM 25A	21	0	18.00±1.41	72.00±5.66	15.67±1.25	68.12±5.42
	NHID^® 25A	7	0	22.67±1.25	90.67±4.99	20.67±1.25	89.86±5.42
	VersaPlex^TM 27PY	2	0	26.33±0.47	97.53±1.75	22.33±0.47	97.10±2.05
	Verifiler^TM Plus	4	0	23.67±0.47	94.67±1.89	21.67±0.47	94.20±2.05
12.5	Identifiler^TM Plus	15	0	11.00±0.82	68.75±5.10	10.67±0.94	71.11±6.26
	AGCU EX28CS	41	1	14.33±2.05	51.19±7.34	12.67±3.30	55.07±14.34
	FastDirect^TM 25A	24	1	17.00±2.16	68.00±8.64	15.33±1.70	66.67±7.39
	NHID^® 25A	29	2	15.33±1.70	61.33±6.80	13.67±1.89	59.42±8.20
	VersaPlex^TM 27PY	32	2	16.33±1.70	60.49±6.30	13.67±0.47	59.42±2.05
	Verifiler^TM Plus	25	2	16.67±0.47	66.67±1.89	16.67±0.47	72.46±2.05

模板质量/pg	试剂盒	等位基因基因座数/个		基因座		常染色体STR
模板质量/pg	试剂盒	缺失	插入	平均检出数/个	平均检出率/%	平均检出数/个	平均检出率/%
50	Identifiler^TM Plus	4	0	14.67±0.47	91.67±2.95	13.33±0.47	88.89±3.14
	AGCU EX28CS	6	2	26.00±1.41	92.86±5.05	21.33±1.25	92.75±5.42
	FastDirect^TM 25A	0	0	25.00±0.00	100.00±0.00	23.00±0.00	100.00±0.00
	NHID^® 25A	0	0	25.00±0.00	100.00±0.00	23.00±0.00	100.00±0.00
	VersaPlex^TM 27PY	2	0	26.33±0.47	97.53±1.75	22.33±0.47	97.10±2.05
	Verifiler^TM Plus	0	0	25.00±0.00	100.00±0.00	23.00±0.00	100.00±0.00
25	Identifiler^TM Plus	14	4	11.00±2.16	68.75±13.50	10.33±1.70	68.59±11.33
	AGCU EX28CS	25	1	19.67±2.87	70.24±10.24	15.67±0.47	68.12±2.05
	FastDirect^TM 25A	21	0	18.00±1.41	72.00±5.66	15.67±1.25	68.12±5.42
	NHID^® 25A	7	0	22.67±1.25	90.67±4.99	20.67±1.25	89.86±5.42
	VersaPlex^TM 27PY	2	0	26.33±0.47	97.53±1.75	22.33±0.47	97.10±2.05
	Verifiler^TM Plus	4	0	23.67±0.47	94.67±1.89	21.67±0.47	94.20±2.05
12.5	Identifiler^TM Plus	15	0	11.00±0.82	68.75±5.10	10.67±0.94	71.11±6.26
	AGCU EX28CS	41	1	14.33±2.05	51.19±7.34	12.67±3.30	55.07±14.34
	FastDirect^TM 25A	24	1	17.00±2.16	68.00±8.64	15.33±1.70	66.67±7.39
	NHID^® 25A	29	2	15.33±1.70	61.33±6.80	13.67±1.89	59.42±8.20
	VersaPlex^TM 27PY	32	2	16.33±1.70	60.49±6.30	13.67±0.47	59.42±2.05
	Verifiler^TM Plus	25	2	16.67±0.47	66.67±1.89	16.67±0.47	72.46±2.05

模板质量/pg	试剂盒（等位基因峰高中位数，单位RFU）						H值	P值
模板质量/pg	Identifiler^TM Plus	AGCU EX28CS	FastDirect^TM 25A	NHID^® 25A	VersaPlex^TM 27PY	Verifiler^TM Plus	H值	P值
200	3 184.500	2 025.250^1）	4 949.000^1）	6 083.000^1）	2 444.000	14 274.000^1）	327.272	0.001
100	1 414.500	1 033.000^1）	2 534.000^1）	3 482.000^1）	1 153.000	3 896.500^1）	301.317	0.001
50	903.250	638.000	1 107.500	1 656.000^1）	622.500^1）	2 902.500^1）	263.192	0.001
25	397.500	279.750	585.500	644.500^1）	309.000	1 029.000^1）	168.687	0.001
12.5	251.250	126.500	424.500	307.500	223.000	436.500^1）	89.562	0.001