Biomics

Nested polymerase chain reaction (nPCR) is a two-step PCR in which the first round uses an outer primer pair to create an amplicon containing binding sites for an inner primer pair. These inner primers, in turn, create a smaller amplicon. A semi-nested PCR variant also exists that employs only one inner primer paired with one of the outer primers in the second round. Nested PCR is primarily designed to increase reaction sensitivity by increasing the copy number of the target sequence in the first round. Additionally, it ensures the specificity of primer annealing and amplification. The inner primers bind to the first-round amplicon, confirming the correct binding of the outer primers. Different methods of separating the first-round reaction mixture from the inner primers before the second round of amplification are described in detail in this review. The review also discusses variations of nested PCR and the practical applications of this approach to amplify target sequences and analyze amplicons, including the detection of DNA polymorphisms. The use of nPCR for solving non-biological problems is noted. 

PCR, nested PCR, nPCR, semi-nested PCR, s-nPCR, primer, outer primer, inner primer, amplification

1. Abath F.G., Melo F.L., Werkhauser R.P., Montenegro L., Montenegro R., Schindler H.C. Single-tube nested PCR using immobilized internal primers. Biotechniques. 2002. V. 33(6). PP. 1210-1214. doi: 10.2144/02336bm05.

2. Alexander A., Subramanian N., Buxbaum J.N., Jacobson D.R. Drop-in, drop-out allele-specific PCR: a highly sensitive, single-tube method. Mol. Biotechnol. 2004. V. 28(3). PP. 171-174. doi: 10.1385/MB:28:3:171.

3. Alirezaei M., Mosawi S.H., Afgar A., Zarean M., Movahhed T.K., Abbasi V., Fotouhi-Ardakani R. Discrimination of human papillomavirus genotypes using innovative technique nested-high resolution melting. Sci. Rep. 2022. V. 12(1). 13943. doi: 10.1038/s41598-022-14730-9.

4. Allan B., Smuts H., Steyn L.M. Modified reaction tubes for the sequential addition of reagents in PCR assays. Nucleic Acids Res. 1994. V. 22(1). PP. 109-110. doi: 10.1093/nar/22.1.109.

5. Alzahrani A.J., Vallely P.J., McMahon R.F. Development of a novel nested in situ PCR-ISH method for detection of hepatitis C virus RNA in liver tissue. J. Virol. Methods. 2002 V. 99(1-2). PP. 53-61. doi: 10.1016/s0166-0934(01)00383-4.

6. Antal Z., Rascle C., Fevre M., Bruel C. Single oligonucleotide nested PCR: a rapid method for the isolation of genes and their flanking regions from expressed sequence tags. Curr. Genet. 2004. V. 46(4). PP. 240-246. doi: 10.1007/s00294-004-0524-6.

7. Avgeris M., Adamopoulos P.G., Galani A., Xagorari M., Gourgiotis D., Trougakos I.P., Voulgaris N., Dimopoulos M.A., Thomaidis N.S., Scorilas A. Novel Nested-Seq Approach for SARS-CoV-2 RealTime Epidemiology and In-Depth Mutational Profiling in Wastewater. Int. J. Mol. Sci. 2021. V. 22(16). 8498. doi: 10.3390/ijms22168498.

8. Bascunana C.R., Belak K. Detection and identification of mycobacteria in formalin-fixed, paraffin-embedded tissues by nested PCR and restriction enzyme analysis. J. Clin. Microbiol. 1996. V. 34(10). PP. 2351-2355. doi: 10.1128/jcm.34.10.2351-2355.1996.

9. Baymiev An.Kh., Baymiev Al.Kh., Kuluev B.R., Shvets K.Yu., Yamidanov R.S., Matniyazov R.T., Chemeris D.A., Zubov V.V., Alekseev Ya.I., Mavzyutov A.R., Ivanenkov Ya.A., Chemeris A.V. Modern approaches to differentiation of live and dead bacteria using selective amplification of nucleic acids. Microbiology. 2020. V. 89, No. 1. P. 13–27. DOI: 10.1134/S0026261720010038

10. Brisco MJ, Bartley PA, Morley AA. Antisense PCR: A simple and robust method for performing nested single-tube PCR // Anal Biochem. 2011. V.409(2). P.176-182. doi: 10.1016/j.ab.2010.10.030

11. Chemeris A.V., Magdanov E.G., Garafutdinov R.R., Vakhitov V.A. How to exclude the appearance of false-positive results during the polymerase chain reaction? Vestn. biotechnol. fiz.-chem. biol. 2012. V. 8(3). P. 34-45. (In Russian)

12. Chemeris D.A., Garafutdinov R.R., Kuluev A.R., Sakhabutdinova A.R., Kuluev B.R., Chemeris A.V. The diversity of methods for the detection of polymorphic nucleotides in the known SNPs. III. Allele-specific PCR. Biomics. 2022. V.14(1). P. 32-51. DOI: 10.31301/2221-6197.bmcs.2022-2 (In Russian)

13. Chemeris D.A., Kiryanova O.Yu., Gubaydullin I.M., Chemeris A.V. Design of primers for polymerase chain reaction (Brief review of software and databases). Biomics. 2016. V. 8(3). P.215-238. (In Russian)

14. Chen F., Suttle C.A. Nested PCR with three highly degenerate primers for amplification and identification of DNA from related organisms. Biotechniques. 1995. V.18(4). P.609-612.

15. Costa J., Mafra I., Kuchta T., Oliveira M.B. Singletube nested real-time PCR as a new highly sensitive approach to trace hazelnut. J. Agric. Food Chem. 2012. V. 60(33). PP. 8103-8110. doi: 10.1021/jf302898z.

16. Daskou M., Tsakogiannis D., Alexopoulou D.S., Dimitriou T.G., Mossialos D., Amoutzias G.D.,             Kottaridi C., Markoulatos P. A colorimetric IsoPCR for the rapid and sensitive visual detection of highrisk HPV16 in clinical samples with hydroxynaphthol blue. J. Virol. Methods. 2021. V. 290. 114072. doi: 10.1016/j.jviromet.2021.114072.

17. Daskou M., Tsakogiannis D., Dimitriou T.G., Manali M., Apti C., Amoutzias G.D., Mossialos D., Kottaridi
C., Markoulatos P. Α 2-stage, nested-like nucleic acid amplification method (IsoPCR) for the highly sensitive detection of HPV16 and HPV18 DNA. Mol. Cell. Probes. 2019. V. 45. PP. 1-7. doi: 10.1016/j.mcp.2019.03.003.

18. Eltahir Y.M., Dovas C.I., Papanastassopoulou M., Koumbati M., Giadinis N., Verghese-Nikolakaki S.,
Koptopoulos G. Development of a semi-nested PCR using degenerate primers for the generic detection of
small ruminant lentivirus proviral DNA. J. Virol. Methods. 2006. V. 135(2). PP. 240-246. doi: 10.1016/j.jviromet.2006.03.010.

19. Ferguson D.A. Jr., Jiang C., Chi D.S., Laffan J.J., Li C., Thomas E. Evaluation of two string tests for
obtaining gastric juice for culture, nested-PCR detection, and combined single- and double-stranded conformational polymorphism discrimination of Helicobacter pylori. Dig. Dis. Sci. 1999. V. 44(10). PP. 2056-2062. doi: 10.1023/a:1026630620442.

20. Fuehrer H.P., Fally M.A., Habler V.E., Starzengruber P., Swoboda P., Noedl H. Novel nested direct PCR
technique for malaria diagnosis using filter paper samples. J. Clin. Microbiol. 2011. V. 49(4). PP. 1628-1630. doi: 10.1128/JCM.01792-10.

21. Garafutdinov R.R., Baymiev An.Kh., Maleev G.V., Alexeyev Ya.I., Zubov V.V., Chemeris D.A., Kiryanova J.Yu., Gubaydullin I.M., Matniyazov R.T., Sakhabutdinova A.R., Nikonorov Yu.M., Kuluev B.R., Baymiev Al.Kh., Chemeris A.V. Diversity of PCR primers and principles of their design. Biomics. 2019. V.11(1). P. 23 – 70. DOI: 10.31301/2221-6197.bmcs.2019-04 (In Russian)

22. Garafutdinov R.R., Chemeris D.A., Mavzyutov A.R., Akhmetzyanova L.U., Davletkulov T.M., Gubaydullin I.M., Chemeris A.V. LAMP amplification of nucleic acids. I. Two decades of development and improvement. Biomics. 2021. V.13(2). P. 176-226. DOI: 10.31301/2221-6197.bmcs.2021-14 (In Russian)

23. Garafutdinov R.R., Chemeris D.A., Mikhailenko K.I., Zubov V.V., Chemeris A.V. Structure of the products
of classical PCR and the reasons for the plateau of the reaction. Biomics. 2024. V.16(1). P.18-32. DOI: 10.31301/2221-6197.bmcs.2024-3 (In Russian)

24. Garafutdinov R.R., Chemeris D.A., Sakhabutdinova A.R., Kiryanova O.Y., Mikhaylenko C.I., Chemeris A.V. Encoding of non-biological information for its long-term storage in DNA. Biosystems. 2022. V. 215-216. 104664. doi: 10.1016/j.biosystems.2022.104664

25. Garafutdinov R.R., Sakhabutdinova A.R., Alekseev Ya.I., Chemeris A.V. Gender loci in DNA forensics and women's sports. Biomics. 2021. V.13(1). P.54-74. DOI: 10.31301/2221-6197.bmcs.2021-6 (In Russian)

26. Garson J.A., Tedder R.S., Briggs M., Tuke P., Glazebrook J.A., Trute A., Parker D., Barbara J.A., Contreras M., Aloysius S. Detection of hepatitis C viral sequences in blood donations by "nested" polymerase chain reaction and prediction of infectivity. Lancet. 1990. V. 335(8703). PP. 1419-1422. doi: 10.1016/0140-6736(90)91446-h.

27. Gomes A.L., Silva A.M., Cordeiro M.T., Guimaraes G.F., Marques E.T. Jr., Abath F.G. Single-tube nested
PCR using immobilized internal primers for the identification of dengue virus serotypes. J. Virol. Methods. 2007. V. 145(1). PP. 76-79. doi: 10.1016/j.jviromet.2007.05.003.

28. Grace M.B., Buzard G.S., Hughes M.R., GoreLangton R.E. Degradable dUMP outer primers in merged tandem (M/T)-nested PCR: low- and singlecopy DNA target amplification. Anal. Biochem. 1998. V. 263(1). PP. 85-92. doi: 10.1006/abio.1998.2771.

29. Green M.R., Sambrook J. Nested Polymerase Chain Reaction (PCR). Cold Spring Harb. Protoc. 2019. V. 2019(2). doi: 10.1101/pdb.prot095182.

30. Guo X., Zhu Y., Pan Z., Pan H., Li H. Single primer site-specific nested PCR for accurate and rapid genome-walking. J. Microbiol. Methods. 2024. V. 220. 106926. doi: 10.1016/j.mimet.2024.106926.

31. Haff L.A. Improved quantitative PCR using nested primers. PCR Methods Appl. 1994. V. 3(6). PP. 332-
337. doi: 10.1101/gr.3.6.332.

32. Hamprecht K., Mikeler E., Jahn G. Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures. J. Virol. Methods. 1997. V. 69(1-2). PP. 125-135. doi: 10.1016/s0166-0934(97)00148-1.

33. Homan W., van Gorkom T., Kan Y.Y., Hepener J. Characterization of Cryptosporidium parvum in human and animal feces by single-tube nested polymerase chain reaction and restriction analysis. Parasitol. Res. 1999. V. 85(8-9). PP. 707-712. doi: 10.1007/s004360050619.

34. Hornes E., Wasteson Y., Olsvik O. Detection of Escherichia coli heat-stable enterotoxin genes in pig stool specimens by an immobilized, colorimetric, nested polymerase chain reaction. J. Clin. Microbiol. 1991. V. 29(11). PP. 2375-2379. doi: 10.1128/jcm.29.11.2375-2379.1991.

35. Huang X.Q., Cloutier S. Hemi-nested touchdown PCR combined with primer-template mismatch PCR for rapid isolation and sequencing of low molecular weight glutenin subunit gene family from a hexaploid wheat BAC library. BMC Genet. 2007. V. 8. 18. doi: 10.1186/1471-2156-8-18.

36. Kashiwamura S., Yamamoto M., Kameda A., Shiba T., Ohuchi A. Potential for enlarging DNA memory: the validity of experimental operations of scaled-up nested primer molecular memory. Biosystems. 2005. V. 80(1). PP. 99-112. doi: 10.1016/j.biosystems.2004.10.007.

37. Keller M., Naue J., Zengerle R., von Stetten F., Schmidt U. Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment. PLoS One. 2015. V. 10(7). e0131845. doi: 10.1371/journal.pone.0131845.

38. Kemp D.J., Churchill M.J., Smith D.B., Biggs B.A., Foote S.J., Peterson M.G., Samaras N., Deacon N.J.,
Doherty R. Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients. Gene. 1990. V. 94(2). PP. 223-228. doi: 10.1016/0378-1119(90)90391-4.

39. Kemp D.J., Smith D.B., Foote S.J., Samaras N., Peterson M.G. Colorimetric detection of specific DNA segments amplified by polymerase chain reactions. Proc. Natl. Acad. Sci. USA. 1989. V. 86(7). PP. 2423-2427. doi: 10.1073/pnas.86.7.2423.

40. Kim B.J., Lee K.H., Park B.N., Kim S.J., Park E.M., Park Y.G., Bai G.H., Kim S.J., Kook Y.H. Detection of rifampin-resistant Mycobacterium tuberculosis in sputa by nested PCR-linked single-strand conformation polymorphism and DNA sequencing. J. Clin. Microbiol. 2001. V. 39(7). PP. 2610-2617. doi: 10.1128/JCM.39.7.2610-2617.2001.

41. Kuluev B.R., Baymiev An.K., Gerashchenkov G.A., Chemeris D.A., Zubov V.V., Kuluev A.R., Baymiev Al.Kh., Chemeris A.V. Random priming PCR strategies for identification of multilocus DNA polymorphism in eukaryotes. Russ. J. Genet. 2018. V. 54(5). P. 499-513. DOI: 10.1134/S102279541805006X

42. Kusser W.C., Levin D.B., Glickman B.W. Sensitive two-stage PCR of p53 genomic DNA exons 5-9. PCR Methods Appl. 1993. V. 2(3). PP. 250-252. doi: 10.1101/gr.2.3.250.

43. Levinson G., Maddalena A., Palmer F.T., Harton G.L., Bick D.P., Howard-Peebles P.N., Black S.H., Schulman J.D. Improved sizing of fragile X CCG repeats by nested polymerase chain reaction. Am. J. Med. Genet. 1994. V. 51(4). PP. 527-534. doi: 10.1002/ajmg.1320510448.

44. Li J., Liu Y.X., Zhao Z.T. Genotyping of hantaviruses occurring in Linyi, China, by nested RT-PCR combined with single-strand conformation polymorphism analysis. Acta Virol. 2009. V. 53(2). PP. 121-124. doi: 10.4149/av_2009_02_121.

45. Li J.S., Tong S.P., Vitvitski L., Trepo C. Single-step nested polymerase chain reaction for detection of different genotypes of hepatitis C virus. J. Med. Virol. 1995. V. 45(2). PP. 151-155. doi: 10.1002/jmv.1890450207. 46. Li W., Han L., Yu P., Ma C., Wu X., Xu J. Nested PCR-denaturing gradient gel electrophoresis analysis of human skin microbial diversity with age. Microbiol. Res. 2014. V. 169(9-10). PP. 686-692. doi: 10.1016/j.micres.2014.02.008.

47. Lok K.S., Lee P.P.F., Kwok Y.C. et al. Nested PCR in magnetically actuated circular closed-loop PCR microchip system. Microchim. Acta. 2012. V. 177. PP. 111–117. doi: 10.1007/s00604-012-0760-2.

48. Luptakova L., Babelova A., Omelka R., Kolena B., Vondrakova M., Bauerova M. Sex determination of early medieval individuals through nested PCR using a new primer set in the SRY gene. Forensic Sci. Int. 2011. V. 207(1-3). P. 1-5. doi: 10.1016/j.forsciint.2010.08.012.

49. Menschikowski M., Vogel M., Eckey R., Dinnebier G., Jaross W. In situ reverse transcriptase-nested polymerase chain reaction to identify intracellular nucleic acids without the necessity of DNAse pretreatment and hybridisation. Anal. Cell. Pathol. 2001. V. 22(3). PP. 151-158. doi: 10.1155/2001/654016.

50. Molina F.I., Geletka L.M., Jong S.C., Zhang Y. Use of a nested primer pair as control for PCR amplification of ribosomal DNA internal transcribed spacers in fungi. Biotechniques. 1994. V. 16(6). PP. 998-1000, 1002.

51. Moser D.A., Neuberger E.W., Simon P. A quick onetube nested PCR-protocol for EPO transgene detection. Drug Test Anal. 2012. V. 4(11). PP. 870-875. doi: 10.1002/dta.1348.

52. Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 1986. V. 51. Pt 1. PP. 263-273. doi: 10.1101/sqb.1986.051.01.032.

53. Mullis K.B., Faloona F.A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987. V. 155. PP. 335-350. doi: 10.1016/0076-6879(87)55023-6.

54. Olmos A., Cambra M., Esteban O., Gorris M.T., Terrada E. New device and method for capture,  reverse transcription and nested PCR in a single closed-tube. Nucleic Acids Res. 1999. V. 27(6). PP. 1564-1565. doi: 10.1093/nar/27.6.1564.

55. Park D.J. 3' RACE LaNe: a simple and rapid fully nested PCR method to determine 3'-terminal cDNA sequence. Biotechniques. 2004. V. 36(4). PP. 586-588. doi: 10.2144/04364BM04.

56. Pecharatana S., Pickett M.A., Watt P.J., Ward M.E. Genotyping ocular strains of Chlamydia trachomatis
by single-tube nested PCR. PCR Methods Appl. 1993. V. 3(3). PP. 200-204. doi: 10.1101/gr.3.3.200.

57. Perrott P., Smith G., Ristovski Z., Harding R., Hargreaves M. A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies. J. Appl. Microbiol. 2009. V. 106(5). PP. 1438-1447. doi: 10.1111/j.1365-2672.2008.04119.x.

58. Porter-Jordan K., Rosenberg E.I., Keiser J.F., Gross J.D., Ross A.M., Nasim S., Garrett C.T. Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. J. Med. Virol.  1990. V. 30(2). PP. 85-91. doi: 10.1002/jmv.1890300202.

59. Poschl B., Waneesorn J., Thekisoe O., Chutipongvivate S., Karanis P. Comparative diagnosis of malaria infections by microscopy, nested PCR, and LAMP in northern Thailand. Am. J. Trop. Med. Hyg. 2010. V. 83(1). PP. 56-60. doi: 10.4269/ajtmh.2010.09-0630.

60. Qin J., Xu M., Zhang Q., Wen X., He S., Zhou Y.,  Liu H., Zhou W. A Nested Asymmetric PCR Melting Curve Assay for One-Step Genotyping of Nondeletional α-Thalassemia Mutations. J. Mol. Diagn. 2020. V. 22(6). PP. 794-800. doi: 10.1016/j.jmoldx.2020.03.002.

61. Ratge D., Scheiblhuber B., Landt O., Berg J., Knabbe C. Two-round rapid-cycle RT-PCR in single closed
capillaries increases the sensitivity of HCV RNA detection and avoids amplicon carry-over. J. Clin. Virol. 2002. V. 24(3). PP. 161-172. doi: 10.1016/s1386-6532(01)00244-x.

62. Raza M.H., Desai S., Aravamudhan S., Zadegan R. An outlook on the current challenges and opportunities in DNA data storage. Biotechnol. Adv. 2023. V. 66. 108155. doi: 10.1016/j.biotechadv.2023.108155.

63. Rusková M., Bučková M., Puškárová A., Cíchová M., Janska V., Achs A., Subr Z., Kuchta T., Pangallo D.
Comparison of ordinary reverse transcription realtime polymerase chain reaction (qRT-PCR) with a newly developed one-step single-tube nested realtime RT-PCR (OSN-qRT-PCR) for sensitive detection of SARS-CoV-2 in wastewater. Environ. Sci. Pollut. Res. Int. 2023. V. 30(42). PP. 95579-95589. doi: 10.1007/s11356-023-29123-2.

64. Salazar M.P., da Costa Lima Suassuna Monteiro J.F., Veloso Carvalho-Silva W.H., Nunes Diniz G.T., Werkhauser R.P., Lapa Montenegro L.M., Schindler H.C. Development and evaluation of a single-tube nested PCR with colorimetric assay for Mycobacterium tuberculosis detection. Biotechniques. 2024. V. 76(6). PP. 235-244. doi: 10.2144/btn-2023-0080.

65. Schierwater B., Metzler D., Kruger K., Streit B. The effects of nested primer binding sites on the reproducibility of PCR: mathematical modeling and computer simulation studies. J. Comput. Biol. 1996. V. 3(2). PP. 235-251. doi: 10.1089/cmb.1996.3.235.

66. Schlayer H.J., Peters T., Preisler S., Fehr J., Gerok W., Rasenack J. Amplification of unknown DNA sequences by sequence-independent nested polymerase chain reaction using a standardized adaptor without specific primers. J. Virol. Methods. 1992. V. 38(3). PP. 333-41. doi: 10.1016/0166-0934(92)90078-r.

67. Sciancalepore A.G., Polini A., Mele E., Girardo S., Cingolani R., Pisignano D. Rapid nested-PCR for tyrosinase gene detection on chip. Biosens. Bioelectron. 2011. V. 26(5). PP. 2711-2715. doi: 10.1016/j.bios.2010.09.008.

68. Sessa R., Schiavoni G., Di Pietro M., Petrucca A., Cipriani P., Puopolo M., Zagaglia C., Fallucca S., Del Piano M. Chlamydia pneumoniae in PBMC: reproducibility of the OMPA nested touchdown PCR. Int. J. Immunopathol. Pharmacol. 2005. V. 18(1). PP. 113-120. doi: 10.1177/039463200501800112.

69. Shatleh-Rantisi D., Tamimi A., Ashhab Y. Improving sensitivity of single tube nested PCR to detect fastidious microorganisms. Heliyon. 2020. V. 6(1). e03246. doi: 10.1016/j.heliyon.2020.e03246.

70. Shaw A.G., Mampuela T.K., Lofiko E.L. et al. Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study. Nat. Microbiol. 2023. V. 8(9). PP. 1634-1640. doi: 10.1038/s41564-023-01453-4.

71. Shimano S., Sambe M., Kasahara Y. Application of nested PCR-DGGE (denaturing gradient gel electrophoresis) for the analysis of ciliate communities in soils. Microbes Environ. 2012. V. 27(2). PP. 136-141. doi: 10.1264/jsme2.me11287.

72. Shuga J., Zeng Y., Novak R., Lan Q., Tang X., Rothman N., Vermeulen R., Li L., Hubbard A., Zhang L., Mathies R.A., Smith M.T. Single molecule quantitation and sequencing of rare translocations using microfluidic nested digital PCR. Nucleic Acids Res. 2013. V. 41(16). e159. doi: 10.1093/nar/gkt613.

73. Smit V.T., Boot A.J., Smits A.M., Fleuren G.J., Cornelisse C.J., Bos J.L. KRAS codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res. 1988. V. 16(16). PP. 7773-7782. doi: 10.1093/nar/16.16.7773.

74. Soe M.J., Rohde M., Mikkelsen J., Warthoe P. IsoPCR: an analytically sensitive, nested, multiplex nucleic acid amplification method. Clin. Chem. 2013. V. 59(2). PP. 436-439. doi: 10.1373/clinchem.2012.193664.

75. Soe M.J., Warthoe P. RT-isoPCR: nested, high multiplex mRNA amplification. Analyst. 2013. V. 138(20). PP. 5871-5874. doi: 10.1039/c3an00803g.

76. Soejima T., Schlitt-Dittrich F., Yoshida S. Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment. Anal. Biochem. 2011. V. 418. № 2. P. 286-289. doi: 10.1016/j.ab.2011.06.033.

77. Song E.Y., Chung H.Y., Joo S.Y., Roh E.Y., Seong M.W., Shin Y., Park M.H. Detection of HLA-DRB1 microchimerism using nested polymerase chain reaction and single-strand conformation polymorphism analysis. Hum. Immunol. 2012. V. 73(3). PP. 291-297. doi: 10.1016/j.humimm.2011.12.016. 78. Song X., Shah Sh., Reif J. Multidimensional Data Organization and Random Access in Large-Scale DNA Storage Systems. bioRxiv. 2019. doi: 10.1101/743369. 

79. Stephensen C.B., Casebolt D.B., Gangopadhyay N.N. Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay. Virus Res. 1999. V. 60(2). PP. 181- 189. doi: 10.1016/s0168-1702(99)00017-9.

80. Sue M.J., Yeap S.K., Omar A.R., Tan S.W. Application of PCR-ELISA in molecular diagnosis. Biomed. Res. Int. 2014. V. 2014. 653014. doi: 10.1155/2014/653014.

81. Tamme R., Camp E., Kortschak R.D., Lardelli M. Nonspecific, nested suppression PCR method for isolation of unknown flanking DNA. Biotechniques. 2000. V. 28(5). PP. 895-899. doi: 10.2144/00285st02.

82. Trka J., Divoky V., Lion T. Prevention of product carry-over by single tube two-round (ST-2R) PCR: application to BCR-ABL analysis in chronic  myelogenous leukemia. Nucleic Acids Res. 1995. V.  23(22). PP. 4736-4737. doi: 10.1093/nar/23.22.4736.

83. Van Loock M., Verminnen K., Messmer T.O., Volckaert G., Goddeeris B.M., Vanrompay D. Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaci in turkeys. BMC Infect. Dis. 2005. V. 5. P. 76. doi: 10.1186/1471-2334-5-76.

84. Vissers E.W., Bodelier P.L., Muyzer G., Laanbroek H.J. A nested PCR approach for improved recovery of archaeal 16S rRNA gene fragments from freshwater samples. FEMS Microbiol. Lett. 2009. V. 298(2). PP. 193-198. doi: 10.1111/j.1574-6968.2009.01718.x.

85. Walsh E.E., Falsey A.R., Swinburne I.A., Formica  M.A. Reverse transcription polymerase chain reaction
(RT-PCR) for diagnosis of respiratory syncytial virus infection in adults: use of a single-tube "hanging droplet" nested PCR. J. Med. Virol. 2001. V. 63(3). PP. 259-263. doi: 10.1002/1096- 9071(200103)63:3<259::AID-JMV1010>3.0.CO;2-X.

86. Wan G., Lim Q.E., Too H.P. High-performance quantification of mature microRNAs by real-time RT PCR using deoxyuridine-incorporated oligonucleotides and hemi-nested primers. RNA. 2010. V. 16(7). PP. 1436-1445. doi: 10.1261/rna.2001610.

87. Wang H.M., Ma W.L., Huang H., Xiao W.W., Wang Y., Zheng W.L. DNA microarray probe preparation by gel isolation nested PCR. J. Biochem. Mol. Biol. 2004. V. 37(3). PP. 356-361. doi: 10.5483/bmbrep.2004.37.3.356.

88. Wang J., Cai K., Zhang R., He X., Shen X., Liu J., Xu J., Qiu F., Lei W., Wang J., Li X., Gao Y., Jiang Y., Xu W., Ma X. Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2. Anal. Chem. 2020. V. 92(13). PP. 9399-9404. doi: 10.1021/acs.analchem.0c01884.

89. Wang J., Zhao L., Sun Z., Li G., Niu P., Li D., Wang L., Zhang Y., Feng Z., Ma X. Development of an innovative one-step nested PCR strategy for virus detection using the LNA technique. Sci. China Life Sci. 2019. V. 62(3). PP. 428-430. doi: 10.1007/s11427-018-9347-1.

90. Wang L.F., Rakela J., Laskus T. Head-to-tail primer tandem repeats generated in hemi-nested PCR. Mol. Cell. Probes. 1997. V. 11(5). PP. 385-387. doi: 10.1006/mcpr.1997.0122.

91. Wang L.T., Smith A., Iacopetta B., Wood D.J., Papadimitriou J.M., Zheng M.H. Nested PCR-SSCP assay for the detection of p53 mutations in paraffin wax embedded bone tumours: improvement of sensitivity and fidelity. Clin. Mol. Pathol. 1996. V. 49(3). M176-8. doi: 10.1136/mp.49.3.m176.

92. Wang Z., Ye S., Li J., Zheng B., Bao M., Ning G. Fusion primer and nested integrated PCR (FPNIPCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotechnol. 2011. V. 11. 109. doi: 10.1186/1472-6750-11-109.

93. Wolff C., Hornschemeyer D., Wolff D., Kleesiek K. Single-tube nested PCR with room-temperature-stable reagents. PCR Methods Appl. 1995. V. 4(6). PP. 376-379. doi: 10.1101/gr.4.6.376. 

94. Xu W.J., Li Q.L., Yao C.J., Wang Z.X., Zhao Y.X., Qiao Z.D. Semi-nested PCR analysis of unknown tags on serial analysis of gene expression. FEBS J. 2008. V. 275(21). PP. 5422-548. doi: 10.1111/j.1742-4658.2008.06671.x.

95. Yeku O., Frohman M.A. Rapid amplification of cDNA ends (RACE). Methods Mol. Biol. 2011. V. 703. PP. 107-122. doi: 10.1007/978-1-59745-248-9_8.

96. Yip C.C., Sridhar S., Leung K.H., Ng A.C., Chan K.H., Chan J.F., Tsang O.T., Hung I.F., Cheng V.C., Yuen K.Y., To K.K. Development and Evaluation of Novel and Highly Sensitive Single-Tube Nested RealTime RT-PCR Assays for SARS-CoV-2 Detection. Int. J. Mol. Sci. 2020. V. 21(16). 5674. doi: 10.3390/ijms21165674.

97. Yourno J. A method for nested PCR with single closed reaction tubes. PCR Methods Appl. 1992. V. 2(1). PP. 60-65. doi: 10.1101/gr.2.1.60.

98. Zhang R.Q., Li Z., Li G.X. et al. A highly sensitive one-tube nested quantitative real-time PCR assay for
specific detection of Bordetella pertussis using the LNA technique. Int. J. Infect. Dis. 2020. V. 93. PP. 224-230. doi: 10.1016/j.ijid.2020.01.053.

99. Zimmermann K., Plaimauer B., Schogl D., Mannhalter J.W. Detection of false-negative results in nested primer PCR of proviral HIV-1 DNA. Biotechniques. 1997. V. 23(5). PP. 882-884, 886, 888. doi: 10.2144/97235st04.