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Improved 18S small subunit rDNA primers for problematic nematode amplification

L. K. Carta
L. K. Carta
 and 
S. Li
S. Li
   | Dec 03, 2018
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Published Online: Dec 03, 2018
Page range: 533 - 542
DOI: https://doi.org/10.21307/jofnem-2018-051
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© 2018 L. K. Carta et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Position diagram of 18S primers constructed with pDRAW32 DNA analysis by AcaClone software. http://www.acaclone.com/ Primer pairs in green and blue are older universal primers (Table 2), and those in red were generated in this study (Table 2).
Position diagram of 18S primers constructed with pDRAW32 DNA analysis by AcaClone software. http://www.acaclone.com/ Primer pairs in green and blue are older universal primers (Table 2), and those in red were generated in this study (Table 2).

Figure 2

Comparison of 18S primers by the PCR amplification. (A) G18S4 and D2AR; (B) 18S-CL-F and D2AR; (C) 18S-CL-F3 and D2AR; M: DNA ladder (0.1-4.0kb); 1: Panagrolaimus sp. Idaho; 2: Aphelenchoides bicaudatus, Maryland; 3: Bursaphelenchus sp. MX; 4: Ditylenchus sp. Idaho; NC was the negative control. The PCRs were performed with TaKaRa Ex Taq as described in Materials and Methods and the conditions: 95°C for 5 min, 35X (95°C for 30′, 50°C for 45′, 72°C for 2 min 30′), 72°C for 2 min, 4°C until finish. An approximately 2,900 nt amplicon was generated.
Comparison of 18S primers by the PCR amplification. (A) G18S4 and D2AR; (B) 18S-CL-F and D2AR; (C) 18S-CL-F3 and D2AR; M: DNA ladder (0.1-4.0kb); 1: Panagrolaimus sp. Idaho; 2: Aphelenchoides bicaudatus, Maryland; 3: Bursaphelenchus sp. MX; 4: Ditylenchus sp. Idaho; NC was the negative control. The PCRs were performed with TaKaRa Ex Taq as described in Materials and Methods and the conditions: 95°C for 5 min, 35X (95°C for 30′, 50°C for 45′, 72°C for 2 min 30′), 72°C for 2 min, 4°C until finish. An approximately 2,900 nt amplicon was generated.

18S-CL-F3 (26 nt, Tm = 60°C) 5′-CTTGTCTCAAAGATTAAGCCATGCAT-3′
G18S4 (21 nt, Tm = 55°C) 5′-GCTTGTCTCAAAGATTAAGCC-3′
18S-CL-F (20 nt, Tm = 53°C) 5′-TCAAAGATTAAGCCATGCAT-3′

Taxa tested successfully with our newly designed ribosomal primers or paired with universal ribosomal primers.

Taxon Primer set Amplified rRNA gene (length) Ta (°C) Taq
Acrobeloides sp. LKC52 Pratylenchus penetrans Thailand 18S-CL-F/18S-CL-R1 18S (around 1,700bp) 50–55 Invitrogen™ Platinum™
Aphelenchoides sp. No1 Idaho Aphelenchoides sp. No2 Idaho Panagrolaimus sp. Idaho 18S-CL-F3/81R 18S and ITS (around 2,400bp) 58 TaKaRa EX
Panagrolaimus sp. LKC44, LKC46 LKC47, LKC53, LKC56, PS1162 Poikilolaimus sp. Idaho 18S-CL-F3/D2AR 18S and ITS (around 2,900bp) 58 TaKaRa EX
Pratylenchus neglectus Malad Idaho Pratylenchus penetrans Maryland Pratylenchus agilis Maryland 18S-CL-F3/AB28 or V2R 18S and ITS (around 2,400bp) 59 Phusion
Ditylenchus sp. Idaho 18S-CL-F6/81R 18S and ITS (around 2,000bp) 54 TaKaRa EX
Bursaphelenchus sp. MX Aphelenchoides sp. No1 Idaho Scutellonema bradys Congo Vittatidera zeaphila New York Deladenus Georgia (in egg masses) Deladenus Georgia (in wood) Pratylenchus sp. Cuba 18S-CL-F3/1912R 18S (around 1,000bp) 50 Invitrogen™ Platinum™
Aphelenchoides sp. No1 Idaho Aphelenchoides sp. No2 Idaho Ditylenchus cf. myceliophagus sp. Idaho Ditylenchus dipsaci Diphtherophora sp. Malad, Idaho 18S-CL-F3/530R 18S (530bp) 50 Invitrogen™ Platinum™
Ditylenchus sp. Idaho 18S-CL-F2/1032R ITS and 28S ( around 2,000bp) 54 TaKaRa EX
Pratylenchus neglectus Malad, Idaho ITS-CL-F2/1006R or 1032R ITS and 28S ( around 2,000bp) 54 TaKaRa EX

Primers used for PCR and sequencing.

Primers Direction Sequence (5′-3′) Tm (°C) rRNA gene PCR Sequencing Reference
18S-CL-F F TCAAAGATTAAGCCATGCAT 53 18S √ √ This study
18S-CL-F2 F CTGTGATGCCCTTAGATGTCC 58 18S √ √ This study
18S-CL-F3 F CTTGTCTCAAAGATTAAGCCATGCAT 60 18S, ITS √ √ This study
18S-CL-F6 F TGAGAAATGGCCACTACGTC 57 18S √ √ This study
18S-CL-R1 R ACCTTGTTACGACTTTTGC 54 18S √ √ This study
18S-CL-R2 R GTTGAGTCAAATTAAGCCGCA 57 18S √ This study
18S-CL-R5 R GCGGTGTGTACAAAGGGCAGGGAC 67 18S √ This study
ITS-CL-F2 F ATTACGTCCCTGCCCTTTGTA 59 ITS, 28S √ √ This study
G18S4 F GCTTGTCTCAAAGATTAAGCC 55 18S √ √ Blaxter et al. (1998)
530R R GCGGCTGCTGGCACCACACTT 68 18S √ √ Thomas (2011)
550F F GGCAAGTCTGGTGCCAGCAGCC 68 18S √ √ Thomas (2011)
1912R R TTTACGGTCAGAACTAGGG 54 18S √ √ Holterman et al. (2006)
81R R TTCCTCCGCTAAATGATATGCTTAA 58 ITS √ √ Reverse of 28S-81for (Holterman et al., 2008)
D2AR R ACTTTCCCTCACGGTACTTGT 59 ITS √ √ Reverse of D2A (Nunn, 1992)
AB28 R ATATGCTTAAGTTCAGCGGGT 57 ITS √ √ Joyce et al. (1994)
VRAIN 2R (V2R) R TTTCACTCGCCGTTACTAAGGGAATC 63 ITS √ √ Vrain et al. (1992)
28S-1006rev (1006R) R AGGGGCGAAAGACTAATCGAAC 60 ITS, 28S √ √ Holterman et al. (2008)
28S-1032rev (1032R) R TCGGAAGGAACCAGCTACTA 57 ITS, 28S √ √ Holterman et al. (2008)

Single pair or multiple pairs of primers frequently used for the amplification of near-full length 18S SSU rDNA.

Primers (old name) Direction Position Single pair Size Reference Note
SSU_F_04 (G18S4) F 963-983 G18S4 and 18P 1,723bp Blaxter et al. (1998)
SSU_R_81 (18P) R 2667-2686 Blaxter et al. (1998)
Nem_SSU_F74 F 1006-1026 Nem_SSU_F74 and 18P 1,680bp Blaxter et al. (1998), Donn et al. (2011)
G18S4 and 18S1573R 1,522bp Yaghoubi et al. (2016)
988F F 969-987 998F and 1912R, 962bp Holterman et al. (2006) 1544–1711 bp generated by these three pairs (Holterman et al., 2006)
1096F F 1076-1095 1096F and 1912R 855bp Holterman et al. (2006)
1912R R 1913-1931 Holterman et al. (2006)
1813F F 1795-1812 1813F and 2646R 870bp Holterman et al. (2006)
2646R R 2646-2665 Holterman et al. (2006)
18S965 F 1898-1920 18S965 and 18S1573R 629bp Mullin et al. (2005), Zeng et al. (2012) 2,121bp generated by these three pairs (Zeng et al., 2012)
18S1573R R 2506-2527 Mullin et al. (2005), Zeng et al. (2012)
SSUF07 F 972-990 SSUF07 and SSUR26 889bp Floyd et al. (2002), Zeng et al. (2012)
SSUR26 R 1841-1861 Floyd et al. (2002), Zeng et al. (2012)
18SnF F 1065-1089 18SnF and 18SnR 1,591bp Kanzaki and Futai (2002), Zeng et al. (2012)
18SnR R 2637-2656 Kanzaki and Futai (2002), Zeng et al. (2012)
18S39F (A or 39F) F 972-990 18S39F(39F) and 18S977R 959bp Blaxter et al. (1998), Olson et al. (2017) 1,706bp generated by these two pairs (Olson et al., 2017)
18S977R R 1910-1931 Olson et al. (2017)
18S900F F 1841-1861 18S900F and 18S1713R 833bp Olson et al. (2017)
18S1713R R 2652-2674 Olson et al. (2017)
18SF Cocktail or Remix: F 18SF Cocktail and 530R 527bp Thomas (2011) 1,400–1,727bp generated by these three pairs (Thomas, 2011)
G18S4 F 963-983 Blaxter et al. (1998)
SSU_F_03 F 963-987 Medlin et al. (1988)
18S-82F F 969-988 Lopez-Garcia et al. (2003)
eukF(10) F 935-955 Medlin et al. (1988)
530R R 1470-1490
385F F 1314-1333 385F and 1108R 754bp Thomas (2011)
1108R R 2047-2068
550F F 1467-1488 550F and 18SR Cocktail 1,200bp Thomas (2011)
18SR Cocktail or Remix: R
18P R 2667-2686 Blaxter et al. (1998)
eukR(10) R 2667-2690 Medlin et al. (1988)
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