Fig 1.
SYBR Green melting profile of the 3.2-kb SIV env SGA-amplicon.
(A) Schematic representation of SIV/HIV full env SGA amplicon. The SIV/HIV genomic RNA (top) with a blow-up of env region and the SGA amplicon (bottom) are shown. The relevant splice donor (SD3 for SIV, SD4 for HIV) and splice acceptor (SA7) sites are indicated. The primer pairs used in the first and second run of SGA PCR are represented by grey and black arrowheads respectively. The resulting full env amplicon (3.2-kb for SIV and 2.9-kb for HIV) used for sequencing and phylogenetic analysis is represented. When different from SIV, information relative to HIV is given in parentheses. (B) Gel electrophoresis analysis of the 3.2-kb SIV env SGA-amplicon. 1.2 μl of negative (lane 1) and positive (lane 2) SIV SGA-nested PCR reactions were loaded on a 1.2% agarose gel and stained with fast red to detect the 3.2-kb amplicon (arrow). (C, D) SYBR Green melting analysis of SIV SGA-PCR reactions. 1.2 μl of positive and negative SGA PCR reaction were added to melting reaction mixture containing increasing amounts of SYBR Green and melting run was performed. The resulting melting curves are presented in C and their corresponding melting peaks in D. The shoulder on the lower temperature side is indicated with a red arrow. Yellow, orange and red melting curves/peaks: melting profile of positive SGA reactions with 1, 2 and 4 μl SYBR Green respectively. Light grey, dark grey and Black melting curves/peaks: melting profile of negative reactions with 1, 2 and 4 μl SYBR Green respectively.
Fig 2.
SYBR Green melt profiling of SIV SGA reactions originated from blood plasma sample.
RNA was extracted from the blood plasma of an SIV infected macaque and used for cDNA synthesis. 24 SIV SGA PCR reactions were performed starting from the RT reaction using a working dilution of 1/33. (A) Agarose gel analysis of 24 SGA reactions. 1.2 μl of the 24 SGA reactions were loaded on 1.2% agarose gel. The positive reactions containing the 3.2-kb env amplicon (black arrow) are indicated with a red arrowhead. (B, C) SYBR Green melting analysis of the 24 SGA reactions. Melting reactions were performed using 1.2 μl of the SGA reactions shown in A and 2 μl of SYBR Green in a total volume of 8 μl. The graphs corresponding to the melting curves and the melting peaks are given in B and C respectively. The red curves are corresponding to the 5 positive reactions indicated in A.
Fig 3.
SYBR Green melting profile of SGA reactions originated from cell and tissue samples.
(A,B,C) Gel electrophoresis analysis of SGA reactions originated from cell and tissue samples. 1.2 μl of SIV env SGA PCR products obtained using undiluted cDNAs from body fluids, cells and tissues (A), serial dilutions of inguinal node cDNA (4x serial dilution from 1/10 to 1/640, 6 replicates each dilution) (B) and cDNAs from semen cells at SGA working dilution (24 replicates) (C), all originated from an SIV infected macaque, was analyzed on 1.2% agarose gel. The 3.2-kb full env and 950-bp MS RNA amplicons are indicated with black and open arrows respectively. Arrowheads in A, B and C indicate the samples used for the melting profiles shown in D–I. D–I) SYBR Green melting profile of SGA reactions originated from cell and tissue samples. The melting curves (D–F) and melting peaks (G–I) corresponding to the samples indicated with the colored arrowheads in A, B and C are given in D and G, E and H, F and I respectively. Melting reactions were performed using 1.2 μl of SGA reaction mixture. The same color is used for arrowhead, melting curve and melting peak of a given sample (red: 3.2-kb env amplicon; dark blue: 950-bp MS RNA amplicon; light blue and orange: mix of amplicons from reactions performed at 1/10 and 1/40 cDNA dilutions respectively; grey: negative reaction). The shoulder on lower Temperature side specific for the full env amplicon is indicated with a red arrow. SP: seminal plasma; BP: blood plasma; PBMC: peripheral blood monocytes; SC: semen cells; Ing-LN: Inguinal Lymph node; EP: epididymis.
Fig 4.
Melting analysis of HIV SGA-amplicons.
RNA extracted from Jurkat cells infected with HIV-1 (IIIB strain) for 8 days was used for cDNA synthesis. HIV cDNAs were quantified using QPCR to determine the SGA working dilution. Next, HIV SGA-PCR was performed using RT serial dilution in a range including the SGA working dilution (6 PCR reactions for each dilution). SGA-PCR performed using 20 copies of pNL-4.3 HIV molecular clone was used as a positive control for full env amplicon. (A) Agarose gel analysis of HIV SGA-PCR products. 1.2 μl of each PCR reaction was loaded on a 1.2% agarose gel and stained with GelRed dye. The 2.9-kb full env and 650-bp MS RNA amplicons are indicated with black and open arrows respectively. Arrowheads are used to show examples of amplification profiles (red and orange for the 2.9-kb full env product amplified from Jurkat cDNA and pNL4.3 plasmid respectively; blue for the 650-bp amplicon corresponding to the MS HIV RNA amplification product; green for a mix of full env and spliced RNA amplification products; black and grey for profiles corresponding to negative reactions). (B,C,D) SYBR Green melting analysis of HIV SGA PCR products. SYBR Green melting reactions were performed using 2 μl (B,C) or 4 μl (D) SYBR Green mix reagent and 1.2 μl of the PCR reactions indicated with arrowheads in A and the resulting melting curves and/or melting peaks are shown. The colors used for melting curves correspond to the one used for arrowheads in A. The shoulder on the low temperature side specific for full env amplification product is indicated with a red arrow. (E,F) EvaGreen melting analysis of HIV SGA PCR products. Melting reactions were performed as indicated for B and C using EvaGreen dye instead of SYBR Green.
Fig 5.
96 well scale screening of SIV env SGA-amplicons using SYBR Green or EvaGreen melt profiling.
cDNA synthesized from RNA extracted from semen cells of SIV infected macaque was used to perform 96 SIV SGA PCR reactions in a 96-well plate using a SGA working dilution of 1/34. 1.2 μl of SGA nested reactions was transferred in 96well plates containing SYBR Green or EvaGreen melting reaction mixture and melting run was performed. The resulting melting curve and melting peak profiles of the full plate (top graphs) were analyzed to isolate the 3.2-kb full env amplicon specific melting profile (bottom graphs) and map the corresponding wells on the plate (red outlined plate). The corresponding SGA positive reactions were collected on the nested PCR plate and sent for sequencing.
Fig 6.
Analysis of a 96-well plate SIV SGA SYBR Green melting profile using the “Graph-2steps” method.
From the full plate SIV SGA SYBR Green melting curve profile (1), the curves with higher intensity level corresponding to the presence of an amplification product are selected (step 1). From the corresponding melting peak profile (2), the peaks with a shoulder (red curves) specific for the 3.2-kb full env amplicon are selected (step 2) and the corresponding wells located on the plate (3). The peaks in blue excluded in step 2 correspond to the MS RNA amplicon. This 2 steps graphical method can be used with any fluorescent dye and for both HIV and SIV SGA with most softwares and will require between 3 and 4 minutes for a 96well plate.
Table 1.
Validation of SYBR Green LAMP in a large scale SIV SGA study.
Fig 7.
LAMP screening of HIV full length env-SGA PCR reactions using a 384-well plate.
1.2 μl of 96 HIV env SGA nested reactions were transferred in a 384-well plate containing the melting mix (EvaGreen dye) and melting run was performed on a CFX384 Touch Real time PCR detection system instrument (Bio-Rad). The melting curve and melting peak profiles were similar to the one observed under 96-well format showing differences in fluorescence intensity and/or peak shape between negative reactions (light green curves) and env (red curves), spliced RNA (blue curves) or other amplicon (orange curves) containing reactions (A). Melting profile was analyzed using the fast “Graph-1click” method by selecting the melting peaks corresponding to the negative and unwanted product containing reactions (B) in order to exclude them and thus map the remaining positive reaction profiles on the plate (C).
Table 2.
Characteristics of the different methods used for melting profile analysis.