Differentiation of subtypes B and E of human immunodeficiency virus type 1 by polymerase chain reaction using novel env gene primers

Yagyu, Fumihiro; Ikeda, Yusei; Ariyoshi, Koya; Sugiura, Wataru; Wongkhomthong, Som Arch; Masuda, Michiaki; Ushijima, Hiroshi

doi:10.1016/s0166-0934(01)00415-3

Cited by 8 publications

(11 citation statements)

References 16 publications

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“…The sensitivity and specificity of these assays are satisfactory. Yagyu developed a PCR subtyping method only differentiating subtype B and CRF01-AE, but the specificity of that assay was found to be lacking (23). Recently, a real-time isothermal amplification assay has been developed to distinguish HIV-1 subtypes A, B, C, CRF01-AE, and CRF02-AG (2).…”

Section: Discussionmentioning

confidence: 99%

Simple Subtyping Assay for Human Immunodeficiency Virus Type 1 Subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC

et al. 2004

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After the initial development of a human immunodeficiency virus type 1 (HIV-1) subtype-screening tool by nested multiplex PCR, we further improve it through the redesign of subtype-specific primers based on subtype signature pattern (SSP) analyses and optimization of the PCR conditions. Extracted RNA from plasma samples was used in reverse transcription and the cDNA products were added to the first round PCR, in which universal primers in the gag region were used to detect HIV-1 M group isolates. In the second round of PCR, three pairs of subtype-specific primers, detecting subtypes B, C, and CRF01-AE, were added in one tube. Subtype determination was based on the different size of PCR products on the agarose gel electrophoresis. An additional set of primers detecting only the prevalent recombinant strains CRF07-BC and CRF08-BC was used to discriminate CRF07-and CRF08-BC from pure subtype C. Testing for all kinds of HIV subtype reference strains indicated that this assay was applicable. A panel of 252 HIV-positive samples and 30 HIV-negative samples was further used to evaluate and validate this assay. Compared to the assay of sequence-based phylogenetic analysis, the newly developed assay has an adequate designated subtype sensitivity, 93.2% (69 of 74) for subtype B, 95.1% (117 of 123) for subtype C, 94.0% (47 of 50) for CRF01-AE, and 95.0% (115 of 121) for CRF07-BC and CRF08-BC. Most importantly, the intersubtype specificity of the assay was found to be 100%. The assay specificity was also found to be 100% when used to test 30 HIV-negative samples. The average reproducibility was 96.0% for subtype B, 96.7% for subtype C, and 95.0% for CRF01-AE. We have developed a simple, rapid, and low cost assay for screening subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC in China.

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Section: Discussionmentioning

confidence: 99%

Simple Subtyping Assay for Human Immunodeficiency Virus Type 1 Subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC

et al. 2004

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“…To examine the integrity of the DNA samples, cellular beta-actin gene was amplified as described previously with b-F and b-R primers [Yagyu et al, 2002] (Table II). The PCR products were subjected to 1% agarose gel electrophoresis at 100 V for 30 min and stained with ethidium bromide.…”

Section: Pcr Of Cellular Beta-actinmentioning

confidence: 99%

“…However, these new methods are more difficult and complicated than PCR. Furthermore, the cost of the single PCR method in particular is approximately 10 times less than that of the sequence method, and thus consideration should be given to its preferential use in developing countries in the future [Yagyu et al, 2002].…”

Section: Introductionmentioning

confidence: 99%

Determination of HIV-1 subtypes (A-D, F, G, CRF01_AE) by PCR in the transmembrane region (gp41) with novel primers

et al. 2005

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HIV-1 has a huge genetic diversity. So far, nine subtypes have been isolated, namely, subtypes A, B, C, D, F, G, H, J, and K. Epidemiological study provides information which may help in the development of HIV-1 prevention programs or health policies. In the future, subtyping may also be critical for vaccine development, and an effective anti-viral drug will need to be effective for different subtypes of HIV virus. The analysis of the nucleotide sequence of the v3 region is considered the most reliable method for determining the HIV-1 subtype. However, the procedures for determining the v3 sequences are complicated and time consuming, requiring expensive reagents, equipment, and well-trained personnel. The polymerase chain reaction (PCR) method using subtype-specific primers for HIV-1 subtyping is easier and faster. The objective of this study was to develop subtype-specific primers for subtyping PCR. The specific primers were designed for subtypes A, B, C, D, F, G, and CRF01_AE, and these primers could be applied to assay for various HIV-1 subtypes in the clinical samples. The specific primers were designed for each subtypes in the gp41 region. The result of PCR was compared with the subtypes which was determined by the v3 sequence. The results of subtyping by PCR using the newly designed primers could detect 29 of 33 patients tested, and all matched those obtained by nucleotide sequencing of the env v3 region except for three subjects, which were differentiated as CRF02_AG. The newly designed primers functioned accurately and conclusively. In comparison with PCR as a method for the determination of subtypes, sequence analysis requires better-trained personnel, more expensive reagents, and more equipment and time.

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“…Assays for HIV-1 subtyping include the heteroduplex mobility assay (HMA) (Delwart et al, 1993(Delwart et al, , 1995Bredell et al, 2000;Heyndrickx et al, 2000;Tatt et al, 2000), V3 loop-peptide ELISA (V3-PEIA) (Gaywee et al, 1996;Hoelscher et al, 1998), subtypespecific PCR in vpu, env, and gag region (Chen et al, 2002;Yagyu et al, 2002;Luo et al, 2011), restriction fragment length polymorphism (RFLP) (van Harmelen et al, 1999), and a single nested multiplex PCR (Gomez-Carrillo et al, 2004). A major limiting factor of these tools is that they cannot identify recombinant strains and dual infections accurately because only parts of one or two genes are examined.…”

Section: Introductionmentioning

confidence: 99%

An effective tool for identifying HIV-1 subtypes B, C, CRF01_AE, their recombinant forms, and dual infections in Southeast Asia by the multi-region subtype specific PCR (MSSP) assay

Sakkhachornphop

Kijak

Beyrer

et al. 2015

Journal of Virological Methods

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Differentiation of subtypes B and E of human immunodeficiency virus type 1 by polymerase chain reaction using novel env gene primers

Cited by 8 publications

References 16 publications

Simple Subtyping Assay for Human Immunodeficiency Virus Type 1 Subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC

Simple Subtyping Assay for Human Immunodeficiency Virus Type 1 Subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC

Determination of HIV-1 subtypes (A-D, F, G, CRF01_AE) by PCR in the transmembrane region (gp41) with novel primers

An effective tool for identifying HIV-1 subtypes B, C, CRF01_AE, their recombinant forms, and dual infections in Southeast Asia by the multi-region subtype specific PCR (MSSP) assay

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