key: cord-0723802-cnfl939g authors: Zimmermann, Fee; Urban, Maria; Krüger, Christian; Walter, Mathias; Wölfel, Roman; Zwirglmaier, Katrin title: In vitro evaluation of the effect of mutations in primer binding sites on detection of SARS-CoV-2 by RT-qPCR date: 2021-06-07 journal: bioRxiv DOI: 10.1101/2021.06.07.447338 sha: 63f60854d701681906a4dfc8c954f673e42efc9d doc_id: 723802 cord_uid: cnfl939g A number of RT-qPCR assays for the detection of SARS-CoV-2 have been published and are listed by the WHO as recommended assays. Furthermore, numerous commercial assays with undisclosed primer and probe sequences are on the market. As the SARS-CoV-2 pandemic progresses, the virus accrues mutations, which in some cases – as seen with the B.1.1.7 variant – can outperform and push back other strains of SARS-CoV-2. If mutations occur in primer or probe binding sites, this can impact RT-qPCR results and impede SARS-CoV-2 diagnostics. Here we tested the effect of primer mismatches on RT-qPCR performance in vitro using synthetic mismatch in vitro transcripts. The effects of the mismatches ranged from a shift in ct values from −0.13 to +7.61. Crucially, we found that a mismatch in the forward primer has a more detrimental effect for PCR performance than a mismatch in the reverse primer. Furthermore, we compared the performance of the original Charité RdRP primer set, which has several ambiguities, with a primer version without ambiguities and found that without ambiguities the ct values are ca. 3 ct lower. Finally, we investigated the shift in ct values observed with the Seegene Allplex kit with the B.1.1.7 SARS-CoV-2 variant and found a three-nucleotide mismatch in the forward primer of the N target. . A R S -C o V -2 s t r a i n m u c I M B -1 t o g e n e r a t e t h e i n v i t r o t r a n s c r i p t s o f t h e m i s m a t c h a n d p e r f e c t m a t c h t e m p l a t e s . m u c I M B -1 ( E P I _ I S L _ 4 0 6 8 6 2 ) w a s i s o l a t e d i n J a n u a r y 2 0 2 0 f r o m a p a t i e n t i n B a v a r i a , G e r m a n y , a n d i s p h y l o g e n e t i c a l l y c l o s e t o t h e f i r s t p u b l i s h e d S A R S -C o V -2 s e q u e n c e , W u h a n -H u 1 . F o r i n v e s t i g a t i n g t h e p e r f o r m a n c e o f s e v e r a l c o m m e r c i a w e u s e d s t r a i n m u c I M B -C B ( E P I _ I S L -7 5 5 6 3 9 ) i n c o m p a r i s o n w i t h m u c I M B -1 . m u c I M B -C B w a s i s o l a t e d f r o m a p a t i e n t w i t h a h i s t o r y o f t r a v e l f r o m t h e U K i n D e c e m b e r 2 0 2 t r u c t i o n o f m i s m a t c h t e m p l a t e s I n o r d e r t o g e n e r a t e R N A t e m p l a t e s w i t h t h e r e q u i r e d m i s m a t c h e s i n t h e p r i m e r b i n d i n g s i t e s , w e u s e d p r i m e r s c o n t a i n i n g t h e m i s m a t c h ( o r c o m p l e m e n t b a s e o f t h e m i s m a t c h f o r r e v e r s e p r i m e r s ) ( S u p p l e m e n t a r y t a b l e 1 ) a n d p e r f o r m e d a n R T -P C R w i t h S A R S -C o V -2 m u c I M B -1 s t r a i n ( w h i c h h a s n o m i s m a t c h t o a n y o f t h e P C R a s s a y s t e s t e d h e r e ) . F o r s e v e r a l m i s m a t c h e s w e h a d t o r e l a x t h e a n n e a l i n g t e m p e r a t u r e i n o r d e r t o b e a b l e t o g e n e r a t e a P C R p r o d u c t ( T a b l e 1 ) . B o t h f o r w a r d a n d r e v e r s e p r i m e r s c o n t a i n e d a s h o r t n o n s e n s e s e q u e n c e 5 ' o f t h e p r i m e r s e q u e n c e s o a s t o g e n e r a t e a P C R p r o d u c t t h a t i s s l i g h t l y l o n g e r t h a n t h e t e m p l a t e f o r t h e s u b s e q u e n t e x p e r i m e n t s . T h e p u r p o s e i s t o a v o i d a r t e f a c t s d u e t o p r i m e r s b i n d i n g r i g h t a t t h e e n d o f a t e m p l a t e . T h e f o r w a r d p r i m e r s c a r r i e d t h e T 7 p r o m o t o r a t t h e 5 ' e n d . W e t h e n u s e d i n v i t r o t r a n s c r i p t i o n t o p r o d u c e t h e m i s m a t c h t e m p l a t e s . C o r r e c t ( i . e . n o m i s m a t c h ) t e m p l a t e s w e r e p r o d u c e d i n t h e s a m e w a y . T h e d e t a i l e d p r o t o c o l i s d e s c r i b e d b e l o w . T o g e n e r a t e t h e D N A t e m p l a t e s n e e d e d f o r i n v i t r o t r a n s c r i p t i o n P C R w a s p e r f o r m e d o n a T h e r m o ™ S c i e n t i f i c P i k o ™ T h e r m a l C y c l e r ( T h e r m o F i s h e r S c i e n t i f e r i n s i l i c o a n a l y s i s s h o w e d t h a t f o r t h e r e v e r s e p r i m e r s e q u e n c e o n l y 2 3 r e s p e c t i v e l y 1 5 o u t o f 6 0 0 , 4 4 3 S A R S -C o V -2 g e n o m e s c a r r y a m i s m a t c h t o t h e . n e w p r i m e r s e q u e n c e i n p o s i t i o n s 3 r e s p e c t i v e l y 1 2 . T h e m i s m a t c h e s a r e s p r e a d o v e r e i g h t ( p o s i t i o n 3 ) a n d n i n e ( p o s i t i o n 1 2 ) l i n e a g e s w i t h n o c l e a r c l u s t e r i n a n y o n e l i n e a g e ( T a b l e 3 b ) . N o g e n o m e c a r r i e s b o t h m i s m a t c h e s a t t h e s a m e t i m e . F o r t h e f o r w a r d p r i m e r 4 4 g e n o m e s c a r r y a C a n d 3 6 a G a t p o s i t i o n 5 o f t h e p r i m e r . T S e v e r a l f a c t o r s c a n i n f l u e n c e t h e i m p a c t o f a m i s m a t c h o n P C R a m p l i f i c a t i o n . O n e i n t u i t i v e a n d w e l l d o c u m e n t e d f a c t o r i s t h e p r o x i m i t y o f t h e m i s m a t c h t o t h e 3 ' e n d o f t h e p r i m e r ( B r u e t a l . , i l l o n e l e t a l . , 2 0 2 0 ) , a f t e r n o t i c i n g a p o o r e r p e r f o r m a n c e o f t h e R d R P a s s a y c o m p a r e d t o t h e E a s s a y a n d c o m p a r i n g 1 6 2 3 S A R S -C o V -2 g e n o m e s e q u e n c e s a v a i l a b l e b y M a y 2 0 2 0 , s u g g e s t e d u s i n g a n R ( = A o r G ) , w h i l e t h e E C D C P r i m e r S c a n w e b s i t e l i s t s a n E C D C -c o r r e c t e d v e r s i o n o f t h e p r i m e r w i t h a W ( = A o r T ) . V o g e l s ( V o g e l s , 2 0 2 0 ) a l s o n o t e d t h e p o o r p e r f o r m a n c e o f t h e R d R P a s s a y a n d s t a t e d t h a t 9 9 0 o f 9 9 V a n a e r s c h o t , M . , M a n n , S . A . , W e b b e r , J . T . , K a m m , J . , B e l l , S . M . , B e l l , J . , H o n g , S . N . , N g u y e n , M . P . , C h a n , L . Y . , B h a t t , K . D . , T a n , M . , D e t w e i l e r , A . M . , E s p i n o s a , A . , W u , W . , B a t s o n , J . , D y n e r m a n , D . , C o n s o r t i u m , C . , W a d f o r d , D . A . , P u s c h n i k , A . S . , N e f f , N . , A h y o n g , V . , M i l l e r , S . , A y s c u e , P . , T a t o , C . M . , P a u l , S . , K i s t l e r , A . L . , D e R i s i , J . L . , C r a w f o r d , E . D . , 2 0 2 1 . I d e n t i f i c a t i o n o f a P o l y m o r p h i s m i n t h e N G e n e o f S A R S -C o V -2 T h a t A d v e r s e l y I m p a c t s D e t e c t i o n b y R e v e r s e T r a n s c r e s u l t s u s i n g t h e p u b l i s h e d p r i m e r s w i t h t h e c o r r e c t t e m p l a t e s a n d m i s m a t c h e d t e m p l a t e s . D i l u t i o n s e r i e s w e r e d o n e i n t r i p l i c a t e . S i z e o f t h e s y m b o l s r e p r e s e n t s o n e ( s m a 1 T a b l e 4 . T r i p l e m i s m a t c h i n t h e S e e g e n e A l l p l e x N -a s s a y i n g e n o m e p o s i t i o n 2 8 2 8 0 -2 8 2 S u p p l e m e n t a r y T a b l e . 1 . P r i m e r a n d p r o b e s e q u e n c e s u s e d i n t h i s s t u d y . U n d e r l i n e d = T 7 p r o m o t o r , i t a l i c i z e d = n o n s e n s e s e q u e n c e t o e x t e n d t h e a m p l i c o n , b o l d a n d r e d = m i s m a t c C D C N 1 2 0 1 9 -n C o V _ N 1 -F G A C C C C A A A A T C A G C G A A A T ( U S -C D C , 2 0 2 0 ) 2 0 1 9 -n C o V _ N 1 -R T C T G G T T A C T G C C A G T T G A A T C T G ( U S -C D C , 2 0 2 G T G C A T A A T T G G C T A C G T C G T G A T G T A G C A C G A T T G C A G C A T T G u s e d t o g e n e r a t e c o r r e c t a n d m i s m a t c h t e m p l a t e C D C N 3 M M F 8 T A A T A C G A C T C A C T A T G T G C A T A A T T G G C T A C G T C G T G A u s e d t o g e n e r a t e c o r r e c t a n d m i s m a t c h n e u T G G C A C C T G T G T A G G T C A A C t e m p l a t e J a p a n N M M F 2 0 T A A T A C G A C T C A C T A T A G G G A G A A C G T T G G C C A A A A T T T T G G G G A C C A G G A A T u s e d t o g e n e r a t e m i s m a t c h t e m p l a t e , m i s m a t c h i n p o s i t i o n 2 0 f r o m 5 ' e n d J a p a n N -n e w M M R 1 7 A C G T G C A T A A T T G G C T A C G T C G T G A T G G C A C C T G T G T A G G T G A A C u s e d t o g e n e r a t e m i s m a t c h t e m p l a United Kingdom 2.0%, Israel 1.0%, Singapore 1 United States of America 97.0%, Canada 2.0%, Denmark 0.0%, United Kingdom 0.0% United States of America 98.0%, Mexico 1.0%, Canada 0.0%, Switzerland 0.0%, South United States of America 0.0% United States of America 26.0%, United Kingdom 21.0%, Bangladesh 6.0%, Canada 5.0%, Germany 5 Reassigned in the current tree United States of America 14 United States of America 26.0%, United Kingdom 21.0%, Bangladesh 6.0%, Canada 5.0%, Germany 5 Reassigned in the current tree TGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGAT MN908947.3(WuhanHu1) CTAAAATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGAT EPI_ISL_755639(B1.1.7) CTAA-ATGTCTCTAAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGAT