Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI

Table 4 Calculation of the melting point confidence interval for each variant group based on real-time RT-PCR HRM curve analysis using LR3.HRM4 or LR3.HRM6 primer pairs ^a

Variant group	2.5^thpercentile^b	97.5^thpercentile^c	Interquartile range (IQR) (75%-25%)^d	Number of outliers (>±1.5xIQR)	Shapiro- Wilk test of normality (p)^e	Melting point interval without overlaps
								Confidence (%)
LR3.HRM4
Group I	83.22	84.08	0.43	0.00	0.103^e	83.20	83.70	67.74
Group II	83.22	84.18	0.45	0.00	0.000	83.15	84.56	100
Group III	84.91	85.65	0.13	6.00	0.000	84.57	85.64	95.89
Group VI	85.35	86.28	0.15	12.00	0.000	85.65	86.37	92.96
LR3.HRM6
Group I	84.79	85.39	0.09	7.00	0.002	84.78	85.69	100
Group II	86.01	86.78	0.42	0.00	0.000	85.70	86.90	100

^aThe data generated for each variant group was tested for normality in order to calculate the largest interval with the highest confidence without overlaps between variant groups. These intervals are indicated in bold. Intervals where all data points (100% confidence) fell within the maximum range, the limits were adjusted to the 2.5^th to 97.5^th percentile to incorporate a margin of error to ensure accurate classification.
^b2.5^th percentile is the melting point temperature where 2.5% of data points is less than or equal to that temperature.
^c97.5^th percentile is the melting point temperature where 2.5% of data points is greater than or equal to that temperature.
^dInterquartile range is the interval where the middle 50% of melting point temperatures can be expected.
^eAssume a normal distribution if p > 0.05, meaning approximately 95% of melting point temperatures of the variant group will be within ±1.96 standard deviations of the mean.

ISSN: 1743-422X