Establishment and application of indirect-ELISA for detection of Mycoplasma parvum based on mp-tGAPDH

doi:10.3969/j.issn.1004-1524.2021.05.05

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (5): 809-815.DOI: 10.3969/j.issn.1004-1524.2021.05.05

• Animal Science • Previous Articles Next Articles

Establishment and application of indirect-ELISA for detection of Mycoplasma parvum based on mp-tGAPDH

FU Yuan(), SHI Tuanyuan, SUN Hongchao

Institute of Animal Husbandry and Veterinary, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2020-10-10 Online:2021-05-25 Published:2021-05-25

Abstract

Abstract:

Hemotropic mycoplasma(HM) resides on the surface of red blood cells and bone marrow of pigs, causing infectious anemia and harm to the pig production. In recent years, three HMs have been found in China, including Mycoplasma suis(M. suis), M. parvum and Candidate Mycoplasma hemosuis(CMh). M. parvum is the predominant hemoplasma strain of the pig populations in Zhejiang and Hainan Province. Currently, there was only M. suis serum antibody detection method was reported. According to the sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of M. parvum ZJ9330 strain, a 404 bp partial fragment was amplified and expressed in E. coli Transetta DE3. The 15 ku truncated protein mp-tGAPDH was purified. Western-blot showed that mp-tGAPDH had higher reactivity with mouse anti mp-tGAPDH serum. The mp-tGAPDH applied as coating antigen, an indirect ELISA method for detecting M. parvum antibody in swine serum was established. The indirect ELISA using mp-tGAPDH exhibited good specificity and no cross-reactivity with porcine circovirus, classical swine fever, pseudorabies, porcine reproductive and respiratory syndrome and Toxoplasma gondii positive serum. Intra-and inter-assay variability was less than 18.4%, showing a high degree of repeatability. The antibody positive rate of M. parvum was 36.69% (91/248) in 248 detected serum samples from 5 pig farms in Zhejiang Province by ELISA. The results showed that indirect ELISA using mp-tGAPDH provides a rapid and simple serological diagnostic tool for the detection of antibodies against M. parvum.

Key words: Hemotropic mycoplasma, Mycoplasma parvum, glyceraldehyde-3-phosphate dehydrogenase, ELISA

CLC Number:

S858

FU Yuan, SHI Tuanyuan, SUN Hongchao. Establishment and application of indirect-ELISA for detection of Mycoplasma parvum based on mp-tGAPDH[J]. Acta Agriculturae Zhejiangensis, 2021, 33(5): 809-815.

Figures/Tables 11

References 13

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指标 Index	脱脂乳Skim milk			BSA
指标 Index	2.5%	5.0%	7.5%	1%	2%	3%
P	0.774	0.494	0.490	1.321	1.225	1.167
N	0.387	0.270	0.223	0.866	0.741	0.663
P/N	2.002	1.832	2.194	1.526	1.653	1.760

指标 Index	脱脂乳Skim milk			BSA
指标 Index	2.5%	5.0%	7.5%	1%	2%	3%
P	0.774	0.494	0.490	1.321	1.225	1.167
N	0.387	0.270	0.223	0.866	0.741	0.663
P/N	2.002	1.832	2.194	1.526	1.653	1.760

指标 Index	5.0%脱脂乳Skim milk			7.5%脱脂乳Skim milk
指标 Index	0.5 h	1.0 h	1.5 h	0.5 h	1.0 h	1.5 h
P	0.781	0.571	0.637	0.548	0.509	0.657
N	0.387	0.278	0.246	0.236	0.231	0.32
P/N	2.019	2.049	2.586	2.322	2.204	2.057

指标 Index	5.0%脱脂乳Skim milk			7.5%脱脂乳Skim milk
指标 Index	0.5 h	1.0 h	1.5 h	0.5 h	1.0 h	1.5 h
P	0.781	0.571	0.637	0.548	0.509	0.657
N	0.387	0.278	0.246	0.236	0.231	0.32
P/N	2.019	2.049	2.586	2.322	2.204	2.057

指标 Index	血清不同作用时间Different serum reaction time
指标 Index	0.5 h	1.0 h	1.5 h
P	0.413	0.627	0.532
N	0.196	0.272	0.255
P/N	2.103	2.303	2.086

Establishment and application of indirect-ELISA for detection of Mycoplasma parvum based on mp-tGAPDH

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指标 Index	不同二抗浓度Different secondary antibody dilution
指标 Index	1:7500	1:10000	1:12500
P	0.497	0.574	0.609
N	0.196	0.183	0.193
P/N	2.540	3.131	3.160

指标 Index	不同二抗作用时间 Different secondary antibody incubating time
指标 Index	30 min	45 min	60 min
P	0.480	0.627	0.666
N	0.155	0.208	0.202
P/N	3.099	3.020	3.302

阳性血清 Positive serum	D₄₅₀	结果判定Result
M. parvum	1.043	+
PCV2	0.191	-
CSFV	0.238	-
PRV	0.297	-
PRRSV	0.076	-
TG	0.078	-
M. suis	0.114	-
CMh	0.533	+

抗原批号 Batch of antigen	血清mp-tGAPDH ELISA重复检测的D₄₅₀值 D₄₅₀ values of mp-tGAPDH ELISA at duplicate detection
抗原批号 Batch of antigen	1	2	3	4	5	6	7
0707	0.946	0.675	0.604	0.607	0.224	0.209	0.151
0707	0.863	0.600	0.560	0.557	0.236	0.190	0.142
0707	0.965	0.659	0.633	0.580	0.231	0.185	0.130
$\bar{X}$	0.905	0.638	0.582	0.582	0.230	0.200	0.147
SD	0.059	0.053	0.031	0.035	0.009	0.013	0.006
CV/%	6.5	8.4	5.3	6.1	3.8	6.7	4.1

抗原批号 Batch of antigen	血清mp-tGAPDH ELISA重复检测的D₄₅₀值 D₄₅₀ values of mp-tGAPDH ELISA at duplicate detection
抗原批号 Batch of antigen	1	2	3	4	5	6
0408	0.612	0.511	0.951	0.161	0.125	0.054
0413	0.508	0.391	0.816	0.128	0.096	0.052
0423	0.500	0.428	0.655	0.127	0.101	0.057
$\bar{X}$	0.540	0.443	0.807	0.139	0.107	0.054
SD	0.062	0.061	0.148	0.019	0.015	0.003
CV/%	11.6	13.8	18.4	13.8	14.3	4.7

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