Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (7): 1458-1468.doi: 10.3864/j.issn.0578-1752.2022.07.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Establishment and Application of PCR Assay for Mycoplasma Contamination in Cell Culture and Live Virus Vaccine

GENG RenHao1,2(),LIU Bo1,WANG Fang1,LUO YuFeng1,QU HongFei1,FAN XueZheng1,QIN YuMing1,DING JiaBo1,XU GuanLong1,SHEN QingChun1(),QIN AiJian2()   

  1. 1China Institute of Veterinary Drug Control, Beijing 100081
    2College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2021-02-08 Accepted:2021-06-21 Online:2022-04-01 Published:2022-04-18
  • Contact: QingChun SHEN,AiJian QIN E-mail:18705279737@163.com;sqcwlx@sina.com;aijian@yzu.edu.cn

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Abstract:

【Objective】Mycoplasma contamination is prone to occur in biological research and productions. Aimed at the shortage of time-effectiveness and sensitivity of current Pharmacopoeia detection methods, a simple, rapid, specific and sensitive PCR method for mycoplasma was established. 【Method】All the sequences of mycoplasmas were extracted from the datasets file SILVA_123_ SSURef from SILVA database, which contained aligned small (16S/18S, SSU) ribosomal RNA (rRNA) sequences for all three domains of life (Bacteria, Archaea and Eukarya). 181 16S rRNA unique sequences of Mycoplasma (including 139 classified species and 42 unclassified Mycoplasma) were obtained by deduplication of the sequences. After alignment, the hypervariable regions V6 to V9 were selected as the detection target regions, and the detection primers were designed to establish a universal PCR method for mycoplasma detection. 12 kinds of mycoplasmas or 2 kinds of acholeplasmas were selected to verify the detection range of the PCR method; 6 kinds of passage cells from different animal and 3 kinds of common bacteria were selected for specific verification; 5 kinds of mycoplasmas and a kind of acholeplasma were selected for sensitivity test. In order to evaluate the performance of the assay in clinical application, 17 batches of animal live virus vaccines ( for 6 kinds of animals) and 24 samples of 8 kinds of cell cultures were subjected to test, which were compared with the classical culture method for mycoplasmas. 【Result】A general PCR method for mycoplasma was established. The detection primers were composed of 2 upstream primers (5'-GCAAARCTATRGARAYA TAGYVGAG-3' and 5'- GCAAAGGCTTAGAAATAAGTTCGGAG-3') and a downstream primer (5'- CCARCTCYCATRGTKTGA CGG - 3'), and the mixing ratio of the primers was 3:1:4. The optimal annealing temperature was 56℃. The PCR method was used to detect 14 mycoplasma species, and the results showed that 396 bp-413 bp specific bands had been amplified, which indicated that the detection range of the PCR method satisfied the detection requirements; No specific bands were amplified from 6 kinds of animal cells and 3 kinds of common bacteria with the PCR assay. The results of sensitivity test showed that the detection limit of the PCR method was 20-200 CCU, and the corresponding nucleic acid was 1.5-15.0 pg. The detection results of 17 batches of live virus vaccines and 24 cell samples were almost consistent with those of culture method, which indicated that the PCR method established in this study was consistent with culture method well, and was more sensitive. 【Conclusion】The PCR assay established in this study was specific, sensitive, easy and fast, suitable for rapid detection of mycoplasma contamination in cells and live virus vaccine.

Key words: cell, live virus vaccine, Mycoplasma, 16S rRNA, PCR

Fig. 1

Results of 16S rRNA gene sequence alignment and the binding locations of Mycoplasma PCR primers"

Fig. 2

The result of primer verification for mycoplasma detection PCR 1: DL2000Marker; 2: Positive control; 3: Negative control; 4: M.hyorhinis(CVCC361); 5: M.synoviae(CVCC385); 6: M.Galliscepticum(CVCC353); 7: M.bovis XBY01 strain; 8: M.hyopneumoniae(CVCC4049); 9: M.iowae(CVCC364); 10: M.hominis HB1 strain; 11:M.anatis BSJ52 strain; 12: M. ovis(CVCC380); 13: M.iners(CVCC363); 14: M.arginini(CVCC346); 15: A.laidlawii(CVCC2406); 16: M.orale(CVCC379); 17: A.axanthum 338 strain; 18: E.coli DH5α; 19: Marc145 cell"

Fig. 3

Optimization results of the PCR assay for Mycoplasma detection a to f: The PCR results with the annealing temperature of 53℃, 54℃, 55℃, 56℃, 57℃ and 58℃, respectively. M: DL2000 Marker; 1: M. galliscepticum; 2: M. synoviae; 3: M.hyorhinis; 4: E.coli; 5: Marc145 cell"

Fig. 4

Specificity of the PCR assay for Mycoplasma detection 1: DL2000 Marker; 2: Positive control; 3: Negative control; 4: MDBK cell; 5: CEF cell; 6: Sf9 cell; 7: Marc145 cell; 8: ST cell; 9: SP2/0 cell; 10: E.coli DH5α; 11: Salmonella CVCC2139; 12: C.perfringens CVCC1125"

Fig. 5

Sequencing result of the PCR production of the positive control"

Fig. 6

Sensitivity of the PCR assay for Mycoplasma detection 1,8,15:DL2000 Marker; Figure a, 2-7: M. galliscepticum deluted to 10-1- -6; 9-14: M.hyorhinis deluted to 10-1- -6; 16-21: M. iowae deluted to 10-1- -6 . Figure b, 2-7: M.arginini deluted to 10-1- -6; 9-14: M.orale deluted to 10-1- -6; 16-21: A.laidlawii deluted to 10-1- -6"

Table 1

The results of the veterinary live vaccines Mycoplasma detection with the PCR assay and traditional culture method"

类别
category		 生物制品名称
Biological products		 原材料
Materials		 阳性结果数Number of positive samples
PCR检测
PCR detection		 培养法检测Cultivationa
细胞
Cells		 健康细胞8株（种）
8 strains(8 species) of healthy cell		 / 0/8 0/8
待检细胞16株（8种）
16 strains(8 species) of cell samples		 / 6/16 4/16（6/16）
动物用病毒
活疫苗
Live vaccines for animals		 鸡用病毒活疫苗6批（品种）
6 batches (species) of live vaccines for chicken		 鸡胚、CEF等
Chicken embryos, CEF, etc.		 1/6 0/6
鸭用病毒活疫苗2批（品种）
2 batches (species) of live vaccines for duck		 鸭胚
Duck embryos		 0/2 0/2
鹅用病毒活疫苗1批（品种）
1 batch (species) of live vaccine for geese		 鸭胚
Duck embryos		 0/1 0/1
猪用病毒活疫苗5批（品种）
3 batches (species) of live vaccines for swine		 Vero、PK15、Marc145、ST等
Vero, PK15, Marc145, ST, etc.		 0/5 0/5
犬用病毒活疫苗2批（品种）
2 batches (species) of live vaccines for dog		 鸡胚、Vero、MDCK、PK15等
Chicken embryos, Vero,MDCK, PK15, etc.		 0/2 0/2
草鱼用病毒活疫苗1批（品种）
1 batch (species) of live vaccines for grass carp		 草鱼胚胎细胞
Embryonic cell of grass carp		 0/1 0/1
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