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Table 2 Description of primers used in PCR and quantitative PCR

From: A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples

Target gene

Primer name and sequences (5′ ➔ 3′)

Amplicon size (bp)

Thermal program

References

18S rRNA

EuK1A: CTGGTTGATCCTGCCAG

499R: CACCAGACTTGCCCTCYAAT

~500

94 °C × 5 min, 35 cycles of 30 s at 94 °C, 60 s at 55 °C, and 90 s at 72 °C, and a final cycle of 10 min at 72 °C.

[40, 41]

ITS

ITS1: TCCGTAGGTGAACCTGCGG

ITS4: TCCTCCGCTTATTGATATGC

~500

95 °C × 15 min, 35 cycles of 30 s at 95 °C, 30 s at 55 °C, and 90 s at 72 °C, and a final cycle of 10 min at 72 °C.

[42]

β-tubulin (qPCR)

BT107F: AACAACTGGGCIAAGGTYACTACAC

BT261R: ATGAAGAAGTGGAGICGIGGGAA

~450

Initial denaturation 20 s at 95 °C, followed by 40 cycles of 1 s at 95 °C and 30 s at 60 °C.

[43]

16S rRNA

341F: CCTACGGGAGGCAGCAG

R806: GGACTACHVGGGTWTCTAAT

~465

94 °C × 5 min, 35 cycles of 45 s at 94 °C, 45 s at 50 °C, and 60 s at 72 °C, and a final cycle of 10 min at 72 °C.

[44, 45]

cpn60

H279: GAIIIIGCIGGIGAYGGIACIACIAC

H280: YKIYKITCICCRAAICCIGGIGCYTT

H1612: GAIIIIGCIGGYGACGGYACSACSAC

H1613: CGRCGRTCRCCGAAGCCSGGIGCCTT

~578

3 min at 94 °C, 40 cycles of 30 s at 94 °C, followed by a temperature gradient of 1 min at 42 °C, 48 °C, 54 °C, or 60 °C, and 1 min at 72 °C, followed by a final extension of 10 min at 72 °C.

[46]

16S rRNA (qPCR)

341F: CCTACGGGAGGCAGCAG

518R: ATTACCGCGGCTGCTGG

~194

Incubation 2 min at 50 °C. Initial denaturation 20 s at 95 °C, followed by 40 cycles of 1 s at 95 °C and 20 s at 60 °C.

[44]

uidA (qPCR)

784F: GTGTGATATCTACCCGCTTCGC

866R: GAGAACGGTTTGTGGTTAATCAGGA

EC807: FAM-TCGGCATCCGGTCAGTGGCAGT-BHQ1

84

Incubation 2 min at 50 °C. Initial denaturation 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

[47]

g23 (qPCR)

MZIA1bis: GATATTTGIGGIGTTCAGCCIATGA

MZIA6: CGCGGTTGATTTCCAGCATGATTTC

~471

94 °C × 1.5 min, 35 cycles of 45 s at 94 °C, 60 s at 50 °C, and 60 s at 72 °C, and a final cycle of 5 min at 72 °C.

Incubation 2 min at 50 °C. Initial denaturation for 20 s at 95 °C, 40 cycles of 1 s at 95 °C and 30 s at 60 °C.

[20]

RdRp

RdRp1: GGRGAYTACASCIRWTTTGAT

RdRp2: MACCCAACKMCKCTTSARRAA

~450

94 °C × 75 s, 40 cycles of 45 s at 94 °C, 45 s at 50 °C, and 60 s at 72 °C, and a final cycle of 5 min at 72 °C.

[26]