The assay of nPCR, which can be considered a variant of cPCR, requires two rounds of PCR amplification using two sets of primers, commonly called the outer primers and the inner primers [
17]. nPCR is more sensitive and specific than cPCR because the probability is extremely low if the first round amplification produces an erroneous fragment; primer pairing and amplification will occur in the second round amplification using the wrong fragment [
51]. The repetitive sequences of SjR2 and SjCHGCS19 (a new 303-bp sequence from non-long terminal repeat (LTR) retrotransposon) are the common biomarkers in nPCR assays [
73,
74]. The published literature showed that the detection limit of SjR2-nPCR stabilized at fg level with 10fg of minimum limit [
75]. Validated by the schistosome-infected mice model [
76], rabbit model [
74,
77], and domestic animals (goat and buffalo) [
78], the SjR2-nPCR or SjCHGCS19-nPCR could all be used for early diagnosis of schistosomiasis, even light infection, showing positive results at 3 days post-infection through testing sera samples. For samples from humans with chronic schistosomiasis, the detection rate of 230-bp SjR2-nPCR assay was 88.79% (95/107), significantly higher than that of the KK method (69.16%, 74/107) [
79]. The sensitivity of the SjR2-nPCR method established by Zhang et al. using 14 and 28 days post-infection buffalo samples was 92.30% (36/39) and 100% (39/39), while the specificity was 97.60% (41/42) [
78]. Moreover, the SjCHGCS19-nPCR assay demonstrated 97.67% sensitivity for 43 patient serum samples and 96.07% specificity for 51 serum samples from healthy individuals [
74]. In addition, an nPCR assay targeting the 420-bp fragment of the Sjα1 gene, which is a short dispersal element retrotransposon gene with high copy and expression throughout the life cycle of schistosomes, could detect 0.1 fg of gDNA and distinguish the infection status of snail 4h post-infection [
80]. However, the impressive performance of the detection efficacy of nPCR needed more verification.